Compositions and methods for promoting wound healing and tissue regeneration

ABSTRACT

Provided herein are compositions and methods for use in promoting wound healing and tissue regeneration following tissue injury in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/542,151, filed Nov. 14, 2014, now U.S. Pat. No. 9,394,351, which is acontinuation of U.S. application Ser. No. 13/842,506, filed Mar. 15,2013, now U.S. Pat. No. 8,916,515, which is a continuation of U.S.application Ser. No. 13/715,626 filed Dec. 14, 2012, now U.S. Pat. No.8,809,257, which is a continuation of U.S. application Ser. No.12/871,461, filed Aug. 30, 2010, now U.S. Pat. No. 8,357,668, which is adivisional of U.S. application Ser. No. 11/721,529, filed Jun. 12, 2007,now U.S. Pat. No. 7,786,074, which is a national stage entry ofInternational Application No. PCT/US2005/046442, filed Dec. 20, 2005,which claims benefit of U.S. Provisional Application No. 60/638,366,filed Dec. 21, 2004 and U.S. Provisional Application No. 60/671,796,filed Apr. 15, 2005, which are all hereby incorporated herein byreference in their entireties.

ACKNOWLEDGEMENTS

This invention was made with government support under Grant RO-1 HL56728awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Your average kid knows that if a skink lizard looses a tail it willeventually grow another one. Moreover, it is well understood amongchildren and grown-ups who make a habit of studying such things thatmany lower animals are capable of regenerating quite complex structures,including whole limbs and organs following injury. For example, fish areable to grow back a heart after a significant part of the old heart ofthe fish had been sliced away (Poss et al., 2002). This is an astoundingresult when one reflects on how essential the heart is to theminute-to-minute survival of most animals.

However, regeneration of tissue, limbs and organs following injury inpeople is not as straightforward as it is in fish. While human tissuesdamaged by mechanical wounding, disease processes and other causes arecapable of healing, complex tissue structure and function is rarely, ifever wholly restored. Instead, recovery of nearly all tissues frominjury in humans and other higher vertebrates is dominated by theformation of scar tissue. The most familiar example of this is thediscolored and fibrotic scars that linger following the healing of askin cut or graze. Less well appreciated is that formation of glial scartissue following injury to the brain or spinal chord is one of the mainobstacles to restoration of neural function following damage to thecentral nervous system (Silver and Miller J H, 2004). There is currentlyno means of treating or preventing such scarring and promoting theregeneration of complex tissue structure and function following injury.

BRIEF SUMMARY OF THE INVENTION

Provided is an isolated polypeptide comprising a carboxy-terminal aminoacid sequence of an alpha Connexin (also referred to herein as an alphaConnexin carboxy-Terminal (ACT) polypeptide), or a conservative variantthereof.

Provided herein is a method of promoting wound healing following tissueinjury in a subject, comprising administering to the subject one or moreof the herein provided compositions (e.g., polypeptides, nucleic acids,or vectors) in a pharmaceutically acceptable carrier.

Additional advantages of the disclosed method and compositions will beset forth in part in the description which follows, and in part will beunderstood from the description, or may be learned by practice of thedisclosed method and compositions. The advantages of the disclosedmethod and compositions will be realized and attained by means of theelements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of thedisclosed method and compositions and together with the description,serve to explain the principles of the disclosed method andcompositions.

FIG. 1 shows that an alpha Connexin carboxy-Terminal (ACT) polypeptideincreases the extent of Cx43 gap junction formation in cultured neonatalmyocytes. Myocytes from neonatal rat hearts were grown until forming anear-confluent monolayer on a tissue culture dish according to standardprotocols. The cultures were subsequently allowed to culture for afurther 5 days in culture medium comprising (a) 30 μM ACT 1 peptide (SEQID NO:2), (b) 30 μM non-active control peptide (SEQ ID NO:55), or (c)phosphate buffered saline (PBS) containing no ACT peptide or control.Culture media with added peptides or vehicle control was changed every24 hours during the experiment. (a) indicates that ACT peptide greatlyincreased the extent of Cx43 gap junction formation (dots and linesindicated by arrowheads) between myocytes relative to the controlconditions (b) and (c). This increase in Cx43 gap junction formation inresponse to ACT peptide is shared by a number of cell types expressingCx43.

FIG. 2 shows that ACT peptide inhibits proliferation and migration oftransformed fibroblasts (NIH-3T3 cells) injured by a scratch. An NIH-3T3monolayer was pre-treated with ACT 1 peptide (SEQ ID NO:2) for 24 hrs,and “scratch-injured” with a p200 pipette tip. The “scratch injury” wassubsequently allowed to “heal” for 24 hours in the presence of (a, b) 30μM ACT 1 peptide (SEQ ID NO:2), (c, d) 30 μM non-active control peptide(SEQ ID NO: 55), or (e, f) vehicle control solution containing no ACTpeptide or control peptide. The “scratch injury” of ACT peptide-treatedcells remains relatively unhealed after 24 hours (a), with few cells(large arrow) repopulating the area within the initial “scratch injury”edges (i.e., within area marked by the small black arrowheads). Bycontrast, in the control conditions in (c) and (e), large numbers ofcells (large arrows) have repopulated the area within the initial“scratch injury”. The repopulation of the “scratch injury” occurs inpart via migration of the transformed cells crawling into the “scratchinjury” area. Figures (b), (d) and (f) show proliferating cell nuclearantigen (PCNA) immunolabeling of cells in the “scratch injury” or at theinjury edge. ACT peptide treated cells (b) show only low luminosityconsistent with background and non-proliferation. Only in the twocontrol conditions shown in (d) and (f), are brightly labeledproliferating cells seen (white arrows). This indicates that the ACTpeptide has also reduced proliferation of the transformed cells.

FIG. 3 shows quantification of the inhibition of migration by ACTpeptides following injury in an experimental cellular model. NIH-3T3fibroblasts were “scratch injured” and subject to the continuouspresence of 30 μM ACT 1 peptide (SEQ ID NO:2) for 24 hours or thecontrol conditions as outlined in FIG. 2. Figure (a) shows the injuryedge of ACT peptide and non-active peptide-treated control cells at theend of the 24-hour period. The cells have been labeled with fluorescentphalloidin to aid visualization. ACT peptide-treated cells show lowlevels of repopulation of the scratch injury area (white double headedarrows). Figure (b) shows a bar graph of the % area of cellsrepopulating the scratch injury after 24 hours. The reduction of cellsin the injury area in the presence of ACT peptide is dramatic, with ap<0.000001.

FIG. 4 shows that expression of an ACT-peptide-encoding-polynucleotideoperably linked to a promoter in the epithelial cell WB-F344 inhibitsmigration following scratch injury in an experimental cellular model.WB-F344 cells are a transformed rat epithelial cell line derived bytreatment of isolated rat liver cells with a cancer-causing agent (Tsaoet al., 1984; Hayashi et al., 1997; Hayashi et al., 1998; Hayashi etal., 2001). WB-F344 cells were transfected with a cDNA expressionplasmid construct and selected under antibiotic using standard protocolsto generate cell lines that stably expressed anACT-peptide-encoding-polynucleotide (SEQ ID NO:6) operably linked to apromoter sequence or a green fluorescent protein (GFP) polynucleotideoperably linked to a promoter sequence as a control. The polynucleotideencoding the ACT peptide also encoded GFP. As such, expression of theACT peptide could be assayed by standard GFP fluorescence optics on alight microscope. (a) and (b) show high magnification images of GFPfluorescence in WB-F344 cell lines expressing GFP plus the carboxyterminus ACT peptide sequence (a) or GFP alone (b). Near confluentmonolayers of the WB-F344 cell lines were “scratch injured” and allowedto “heal” for 24 hours. Similar to the control cases of the NIH-3T3cells treated with vehicle or non-active control peptide, the controlepithelial cell line expressing GFP repopulated the scratch injury (d).However, in the epithelial cell line that stably expressed theACT-peptide-encoding-polynucleotide operably linked to a promotersequence, there was inhibited repopulation of the scratch injury (c).

FIG. 5 shows that ACT peptide reduces inflammation, improves healing andreduces scarring following incisional skin injury in a neonatal mouse.Neonatal mouse pups were desensitized using hypothermia. A 4 mm longincisional skin injury was made using a scalpel through the entirethickness of the skin (down to the level of the underlying muscle) inthe dorsal mid line between the shoulder blades. 30 μl of a solution of20% pluronic (F-127) gel containing either no (control) or dissolved ACT1 peptide (SEQ ID NO: 2) at a concentration of 60 μM was then applied tothe incisional injuries. Control or ACT peptide containing gel wasapplied subsequently 24 hours after the initial application. No furtherapplication of control and ACT peptide containing gel was made after thesecond application. By 48 hours the ACT peptide treated injury (a) issignificantly more closed, less inflamed, less swollen (note ridges atthe wound edge), and generally more healed in appearance than thecontrol injury that received no ACT peptide (b). These differences ininflammation, swelling and healing between the control and ACT peptideand control treated injury persisted at the 72 (c, d) and 96 (e, f) hourtime points. At 7 days, the ACT peptide wound (g), had a smoother andless scarred appearance than the control peptide-treated injury (h).Note that images of the same injury on the same animal are shown at thedifferent time points during the healing time course.

FIG. 6 shows that ACT peptide reduces inflammation, improves healing andreduces scarring following a large excisional skin injury in adult mice.Anesthetized adult mice had 8 mm wide circular excisional skin injuriesmade by fine surgical scissors down to the underlying muscle in thedorsal mid line between the shoulder blades (i.e., as shown in (a) an(b). The boundary of the injury was demarcated by an 8 mm wide circulartemplate cut in a plastic sheet. 100 μl of a solution of 30% pluronicgel containing either no (control) or dissolved ACT 1 peptide (SEQ IDNO:2) at a concentration of 100 μM was then applied to the excisionalinjuries. Control or ACT peptide containing gel was applied subsequently24 hours after the initial application. No further applications ofcontrol and ACT peptide containing gel were made after the secondapplication. The ACT peptide-treated large excisional injury (a, c e, g,i) closed faster, was less inflamed in appearance, healed faster andscarred less than the control injury that received no ACT peptide (b, d,f, h, j) over the 14 day time course. Indeed, the control injury at 14days still shows a partial scab indicating that acute healing of theinjury was incomplete (j). Note that images of the same injury on thesame animal are shown at the different time points during the healingtime course.

FIG. 7 shows that ACT peptide reduces the density of inflammatory cellsfollowing excisional skin injury in adult mice. Skin biopsies of theentire wound site were taken from some of the mice 24 hours followingthe excisional injury in the experiment described in FIG. 6. Figures (a)and (b) show low magnification survey views of cross-sections from nearthe center of the wound of control and ACT peptide treated injuriesrespectively. The wound edge (marked by the small arrows), bounded byskin of normal histological appearance, can be seen in both cases. Ablack rectangle is placed over the images in (a) and (b) at the lefthand wound edge. The histological structures within these two rectanglesare shown at higher magnification in (c) and (d) for control and ACTpeptide treated tissues, respectively. Of most interest is a“collar-like” tissue of aligned fibrous material (arrowed) projectingfrom basal parts of the injury to or toward the wound edge and exteriorsurface of injury. The aligned fibrous substrate has the appearance ofbeing much more organized in the control injury (d) than in the ACTpeptide treated injury (c). Also, there is a considerably lower densityof inflammatory cells studding the fibrous substrate in the ACTpeptide-treated tissue. This is confirmed in (e) and (f) where regionsof histological section within the black rectangles shown in (c) and (d)are respectively shown at higher magnification. The inflammatory cellsstudding the aligned fibrous substrate include mast cells, neutrophilsand macrophages. These inflammatory cells occur at much higher densityin the control injury than in the ACT peptide treated injury.

FIG. 8 shows that ACT peptide promotes healing, reduces scarring andpromotes regeneration of complex tissue structure following excisionalskin injury in adult mice. At the end of the 14 day period in theexperiment described in FIG. 6, skin biopsies of the entire excisionalinjury were taken and histological sections from these skin samples wereH&E histochemically stained. Figures (a) and (b) show low magnificationsurvey views of cross-sections from near the center of the injury of ACTpeptide and control respectively. The wound edge (marked by the smallarrows), bounded by skin of normal histological appearance, can be seenin both cases. A black rectangle is placed over the images in (a) and(b) near the center of each injury. The histological structures withinthese two rectangles are shown at higher magnification in (c) and (d)for the ACT peptide and control tissues respectively. It is evident thattissue within the ACT peptide treated injury locus has considerably morecomplexity. At the external surface of the ACT treated wound, there is acontinuous layer of epithelial cells indicating that re-epithelizationof the injured surface is complete, albeit that the epithelium is as yetrelatively thin near the center of the wound (c). Regenerating hairfollicles can already be seen differentiating de novo from stem cells inthe new epithelium covering the healed injury (c, small arrows). Bycomparison, re-epithelization of the injury surface is incomplete andthere is no sign of regenerating hair follicles in the epithelium of thecontrol injury (d). Beneath the reformed epithelium of the ACT peptidetreated injured skin, considerable restoration of normal structuralcomplexity is seen, with glandular structures, fibrous and connectivetissues, vascular tissues, muscle and fat cells all in evidence (a, c).As with, the hair follicles this tissue complexity was regenerated bydifferentiation of stem cells. By contrast, in the control injury thewound tissue is completely dominated by a uniform and large plug offibrous scar tissue (b, d), with other complexity of tissue structurenot particularly in evidence within this scar tissue

FIG. 9 shows that ACT peptides reduce inflammation, improve healing andreduce scarring following excisional skin injury in adult mice.Anesthetized adult mice had 2 small (5 mm diameter) excisional skinwounds made by fine surgical scissors on the neck and (upper) back. Theboundaries of the injuries were demarcated by a 5 mm wide circulartemplate cut in a plastic sheet. 50-60 μl of a solution of 20% pluronicgel containing either no (control) or one of the ACT peptides (ACT 2-SEQID NO:1, ACT 1-SEQ ID NO:2, ACT 3-SEQ ID NO:3, ACT 4-SEQ ID NO:4, ACT5-SEQ ID NO:5) dissolved at concentrations of 100 μM were then appliedto the excisional injuries. Control or ACT peptide-containing gel wasapplied subsequently 24 hours after the initial application. No furtherapplications of control and ACT peptide-containing gel were made afterthe second application. It can be noted in the case of ACT 1 (e-h), ACT2 (i-l), ACT 3 (m-p), and ACT 5 (u-x) peptides that excisional injuriesclosed faster, were significantly less inflamed in appearance, healedfaster and scarred less than the control injury that received no ACTpeptide (a-d) over the 240 hour time course (10 days). The ACT 4 peptide(q-t) also showed modest improvement in healing over the control duringthe time course, although less so than other peptides. Note that thesame wound on the same animal is shown at the different time pointsduring the healing time course.

FIG. 10 shows that ACT peptide reduces the number and density of glialscar forming astrocytes following penetration injury of brain in anadult rat. (b) and (c) show low magnification survey views of sectionsof brain tissue (cortex) surrounding hollow fiber membrane (HFM)implants filled with ACT peptide (100 μM) plus vehicle gel (b) orcollagen vehicle gel alone as control (c). In the control tissue (c), ahigh density of immunolabeled GFAP-positive astrocytes is observed nearthe site of injury caused by the HFM. The density of these cells appearsto diminish slightly distal from the injury. By contrast, a much lowerdensity of GFAP-positive astrocytes is observed adjacent the HFM filledwith ACT peptide (b). Indeed, the levels of GFAP positive cells are notdissimilar to those seen in normal uninjured brain tissue. The regionsof tissue within the white rectangles in figures (b) and (c) are shownat higher magnification in (d) and (e) respectively. In the brain injurytreated by ACT peptide (d), it can be seen that GFAP-positive astrocytesare not only less numerous, but are also smaller than those seen in thecontrol injury (e).

FIG. 11 shows that ACT peptide promotes neuronal maintenance andneuronal regeneration following penetration injury of brain in an adultrat. (a) and (b) show low magnification survey views of sections ofbrain tissue (cortex) surrounding HFM implants (implant or injury borderis shown by arrows) filled with control collagen vehicle gel or ACTpeptide plus vehicle gel at 1 week following brain penetration injury.In the control tissue (b), a high density of immunolabeled GFAP-positiveastrocytes and a low density of NeuN immunolabeled neurons are observednear the site of injury caused by the HFM. The density of these cellsappears to diminish and increase, respectively, distal from the HFM. Bycontrast, a much lower density of GFAP-positive astrocytes and highernumbers NeuN immunolabeled neurons are observed proximal (as well asdistal) to the HFM filled with ACT peptide (a). The areas in (a) and (b)proximal to the HFMs are shown at high magnification views in (c) and(b), respectively. Again, in the control tissue (d) a striking increasein the density of GFAP-positive astrocytes and a reduced density ofNeuN-positive neurons is observed compared to ACT peptide treated tissue(c). A complementary pattern is observed near the HFM containing ACTpeptide, with NeuN positive neurons predominating over astrocytes (c).Interestingly, the high magnification view shown in (c) reveals a highfrequency of neurons in the process of fission relative to the control(d).

DETAILED DESCRIPTION OF THE INVENTION

The disclosed method and compositions may be understood more readily byreference to the following detailed description of particularembodiments and the Examples included therein and to the Figures andtheir previous and following description.

Provided is an isolated polypeptide comprising a carboxy-terminal aminoacid sequence of an alpha Connexin (also referred to herein as an alphaConnexin carboxy-Terminal (ACT) polypeptide), or a conservative variantthereof. In one aspect, following tissue injury, the provided ACTpolypeptide reduces inflammation, promotes healing, reduces scarring,increases tensile strength, and promotes complex tissue regeneration. Inanother aspect, the provided polypeptide increases the extent of gapjunctional channel aggregates formed from Connexins.

It is to be understood that the disclosed compositions and methods arenot limited to specific synthetic methods, specific analyticaltechniques, or to particular reagents unless otherwise specified, and,as such, may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed method and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a vector is disclosed and discussed and a numberof vector components including the promoters are discussed, each andevery combination and permutation of promoters and other vectorcomponents and the modifications that are possible are specificallycontemplated unless specifically indicated to the contrary. Thus, if aclass of molecules A, B, and C are disclosed as well as a class ofmolecules D, E, and F and an example of a combination molecule, A-D isdisclosed, then even if each is not individually recited, each isindividually and collectively contemplated. Thus, is this example, eachof the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F arespecifically contemplated and should be considered disclosed fromdisclosure of A, B, and C; D, E, and F; and the example combination A-D.Likewise, any subset or combination of these is also specificallycontemplated and disclosed. Thus, for example, the sub-group of A-E,B-F, and C-E are specifically contemplated and should be considereddisclosed from disclosure of A, B, and C; D, E, and F; and the examplecombination A-D. This concept applies to all aspects of this applicationincluding, but not limited to, steps in methods of making and using thedisclosed compositions. Thus, if there are a variety of additional stepsthat can be performed it is understood that each of these additionalsteps can be performed with any specific embodiment or combination ofembodiments of the disclosed methods, and that each such combination isspecifically contemplated and should be considered disclosed.

A variety of sequences are provided herein and these and others can befound in Genbank at www.pubmed.gov. Those of skill in the art understandhow to resolve sequence discrepancies and differences and to adjust thecompositions and methods relating to a particular sequence to otherrelated sequences. Primers and/or probes can be designed for anysequence given the information disclosed herein and known in the art.

The herein provided polypeptide can be any polypeptide comprising thecarboxy-terminal most amino acids of an alpha Connexin, wherein thepolypeptide does not comprise the full-length alpha Connexin protein.Thus, in one aspect, the provided polypeptide does not comprise thecytoplasmic N-terminal domain of the alpha Connexin. In another aspect,the provided polypeptide does not comprise the two extracellular domainsof the alpha Connexin. In another aspect, the provided polypeptide doesnot comprise the four transmembrane domains of the alpha Connexin. Inanother aspect, the provided polypeptide does not comprise thecytoplasmic loop domain of the alpha Connexin. In another aspect, theprovided polypeptide does not comprise that part of the sequence of thecytoplasmic carboxyl terminal domain of the alpha Connexin proximal tothe fourth transmembrane domain. There is a conserved proline or glycineresidue in alpha Connexins consistently positioned some 17 to 30 aminoacids from the carboxyl terminal-most amino acid (Table 2). For example,for human Cx43 a proline residue at amino acid 363 is positioned 19amino acids back from the carboxyl terminal most isoleucine. In anotherexample, for chick Cx43 a proline residue at amino acid 362 ispositioned 18 amino acids back from the carboxyl terminal-mostisoleucine. In another example, for human Cx45 a glycine residue atamino acid 377 is positioned 19 amino acids back from the carboxylterminal most isoleucine. In another example for rat Cx33, a prolineresidue at amino acid 258 is positioned 28 amino acids back from thecarboxyl terminal most methionine. Thus, in another aspect, the providedpolypeptide does not comprise amino acids proximal to said conservedproline or glycine residue of the alpha Connexin. Thus, the providedpolypeptide can comprise the c-terminal-most 4 to 30 amino acids of thealpha Connexin, including the c-terminal most 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30 amino acids of the alpha Connexin.

The carboxy-terminal most amino acids of an alpha Connexin in theprovided peptides can be flanked by non-alpha Connexin or non-ACTpeptide Connexin amino acids. Examples of the flanking non-alphaConnexin and non-ACT Connexin amino acids are provided herein. Anexample of non-ACT Connexin amino acids are the carboxy-terminal 20 to120 amino acids of human Cx43 (SEQ ID NO: 72). Another example would bethe carboxy-terminal 20 to 120 amino acids of chick Cx43 (SEQ ID NO:73). Another example would be the carboxy-terminal 20 to 120 amino acidsof human Cx45 (SEQ ID NO: 74). Another example would be thecarboxy-terminal 20 to 120 amino acids of chick Cx45 (SEQ ID NO: 75).Another example would be the carboxy-terminal 20 to 120 amino of humanCx37 (SEQ ID NO: 76). Another example would be the carboxy-terminal 20to 120 amino acids of rat Cx33 (SEQ ID NO: 77).

An example of a non-alpha Connexin is the 239 amino acid sequence ofenhanced green fluorescent protein (ACT1 is shown functionally fused toGFP in FIG. 4; SEQ ID NO: 78). In another aspect, given that ACT1 isshown to be functional when fused to the carboxy terminus of the 239amino acid sequence of GFP (e.g., FIG. 4), ACT peptides are expected toretain function when flanked with non-Connexin polypeptides of up to atleast 239 amino acids. Indeed, as long as the ACT sequence is maintainedas the free carboxy terminus of a given polypeptide, and the ACT peptideis able to access its targets. Thus, polypeptides exceeding 239 aminoacids in addition to the ACT peptide can function in reducinginflammation, promoting healing, increasing tensile strength, reducingscarring and promoting tissue regeneration following injury.

Connexins are the sub-unit protein of the gap junction channel which isresponsible for intercellular communication (Goodenough and Paul, 2003).Based on patterns of conservation of nucleotide sequence, the genesencoding Connexin proteins are divided into two families termed thealpha and beta Connexin genes. The carboxy-terminal-most amino acidsequences of alpha Connexins are characterized by multiple distinctiveand conserved features (see Table 2). This conservation of organizationis consistent with the ability of ACT peptides to form distinctive 3Dstructures, interact with multiple partnering proteins, mediateinteractions with lipids and membranes, interact with nucleic acidsincluding DNA, transit and/or block membrane channels and provideconsensus motifs for proteolytic cleavage, protein cross-linking,ADP-ribosylation, glycosylation and phosphorylation. Thus, the providedpolypeptide interacts with a domain of a protein that normally mediatesthe binding of said protein to the carboxy-terminus of an alphaConnexin. For example, nephroblastoma overexpressed protein (NOV)interacts with a Cx43 c-terminal domain (Fu et al., J Biol Chem. 2004279(35):36943-50). It is considered that this and other proteinsinteract with the carboxy-terminus of alpha Connexins and furtherinteract with other proteins forming a macromolecular complex. Thus, theprovided polypeptide can inhibit the operation of a molecular machine,such as, for example, one involved in regulating the aggregation of Cx43gap junction channels.

As used herein, “inhibit,” “inhibiting,” and “inhibition” mean todecrease an activity, response, condition, disease, or other biologicalparameter. This can include, but is not limited to, the complete loss ofactivity, response, condition, or disease. This can also include, forexample, a 10% reduction in the activity, response, condition, ordisease as compared to the native or control level. Thus, the reductioncan be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount ofreduction in between as compared to native or control levels.

The ACT sequence of the provided polypeptide can be from any alphaConnexin. Thus, the alpha Connexin component of the provided polypeptidecan be from a human, murine, bovine, monotrene, marsupial, primate,rodent, cetacean, mammalian, avian, reptilian, amphibian, piscine,chordate, protochordate or other alpha Connexin.

Thus, the provided polypeptide can comprise an ACT of a Connexinselected from the group consisting of mouse Connexin 47, human Connexin47, Human Connexin 46.6, Cow Connexin 46.6, Mouse Connexin 30.2, RatConnexin 30.2, Human Connexin 31.9, Dog Connexin 31.9, Sheep Connexin44, Cow Connexin 44, Rat Connexin 33, Mouse Connexin 33, Human Connexin36, mouse Connexin 36, rat Connexin 36, dog Connexin 36, chick Connexin36, zebrafish Connexin 36, morone Connexin 35, morone Connexin 35,Cynops Connexin 35, Tetraodon Connexin 36, human Connexin 37, chimpConnexin 37, dog Connexin 37, Cricetulus Connexin 37, Mouse Connexin 37,Mesocricetus Connexin 37, Rat Connexin 37, mouse Connexin 39, ratConnexin 39, human Connexin 40.1, Xenopus Connexin 38, ZebrafishConnexin 39.9, Human Connexin 40, Chimp Connexin 40, dog Connexin 40,cow Connexin 40, mouse Connexin 40, rat Connexin 40, Cricetulus Connexin40, Chick Connexin 40, human Connexin 43, Cercopithecus Connexin 43,Oryctolagus Connexin 43, Spermophilus Connexin 43, Cricetulus Connexin43, Phodopus Connexin 43, Rat Connexin 43, Sus Connexin 43, MesocricetusConnexin 43, Mouse Connexin 43, Cavia Connexin 43, Cow Connexin 43,Erinaceus Connexin 43, Chick Connexin 43, Xenopus Connexin 43,Oryctolagus Connexin 43, Cyprinus Connexin 43, Zebrafish Connexin 43,Danio aequipinnatus Connexin 43, Zebrafish Connexin 43.4, ZebrafishConnexin 44.2, Zebrafish Connexin 44.1, human Connexin45, chimp Connexin45, dog Connexin 45, mouse Connexin 45, cow Connexin 45, rat Connexin45, chick Connexin 45, Tetraodon Connexin 45, chick Connexin 45, humanConnexin 46, chimp Connexin 46, mouse Connexin 46, dog Connexin 46, ratConnexin 46, Mesocricetus Connexin 46, Cricetulus Connexin 46, ChickConnexin 56, Zebrafish Connexin 39.9, cow Connexin 49, human Connexin50, chimp Connexin 50, rat Connexin 50, mouse Connexin 50, dog Connexin50, sheep Connexin 49, Mesocricetus Connexin 50, Cricetulus Connexin 50,Chick Connexin 50, human Connexin 59, or other alpha Connexin. Aminoacid sequences for alpha connexins are known in the art and includethose identified in Table 1 by accession number.

TABLE 1 Alpha Connexins Protein Accession No. Protein Accession No.mouse Connexin 47 NP_536702 Phodopus Connexin 43 AAR33085 human Connexin47 AAH89439 Rat Connexin 43 AAH81842 Human Connexin46.6 AAB94511 SusConnexin 43 AAR33087 Cow Connexin 46.6 XP_582393 Mesocricetus Connexin43 AAO61857 Mouse Connexin 30.2 NP_848711 Mouse Connexin 43 AAH55375 RatConnexin 30.2 XP_343966 Cavia Connexin 43 AAU06305 Human Connexin 31.9AAM18801 Cow Connexin 43 NP_776493 Dog Connexin 31.9 XP_548134 ErinaceusConnexin 43 AAR33083 Sheep Connexin 44 AAD56220 Chick Connexin 43AAA53027 Cow Connexin 44 I46053 Xenopus-Connexin 43 NP_988856 RatConnexin 33 P28233 Oryctolagus Connexin 43 AAS89649 Mouse Connexin 33AAR28037 Cyprinus Connexin 43 AAG17938 Human Connexin 36 Q9UKL4Zebrafish Connexin 43 CAH69066 mouse Connexin 36 NP_034420 Danioaequipinnatus Connexin 43 AAC19098 rat Connexin 36 NP_062154 ZebrafishConnexin 43.4 NP_571144 dog Connexin 36 XP_544602 Zebrafish Connexin44.2 AAH45279 chick Connexin 36 NP_989913 Zebrafish Connexin 44.1NP_571884 zebrafish Connexin 36 NP_919401 human Connexin45 I38430 moroneConnexin 35 AAC31884 chimp Connexin45 XP_511557 morone Connexin 35AAC31885 dog Connexin 45 XP_548059 Cynops Connexin 35 BAC22077 mouseConnexin 45 AAH71230 Tetraodon Connexin 36 CAG06428 cow Connexin 45XP_588395 human Connexin 37 I55593 rat Connexin 45 AAN17802 chimpConnexin 37 XP_524658 chick Connexin45 NP_990834 dog Connexin 37XP_539602 Tetraodon Connexin 45 CAF93782 Cricetulus Connexin 37 AAR98615chick Connexin 45.6 I50219 Mouse Connexin 37 AAH56613 human Connexin 46NP_068773 Mesocricetus Connexin37 AAS83433 chimp Connexin 46 XP_522616Rat Connexin37 AAH86576 mouse Connexin 46 NP_058671 mouse Connexin 39NP_694726 dog Connexin 46 XP_543178 rat Connexin 39 AAN17801 ratConnexin 46 NP_077352 human Connexin 40.1 NP_699199 MesocricetusConnexin 46 AAS83437 Xenopus Connexin38 AAH73347 Cricetulus Connexin 46AAS77618 Zebrafish Connexin 39.9 NP_997991 Chick Connexin 56 A45338Human Connexin 40 NP_859054 Zebrafish Connexin 39.9 NP_997991 ChimpConnexin 40 XP_513754 cow Connexin 49 XP_602360 dog Connexin 40XP_540273 human Connexin 50 P48165 cow Connexin 40 XP_587676 chimpConnexin 50 XP_524857 mouse Connexin 40 AAH53054 rat Connexin 50NP_703195 rat Connexin 40 AAH70935 mouse Connexin 50 AAG59880 CricetulusConnexin 40 AAP37454 dog Connexin 50 XP_540274 Chick Connexin 40NP_990835 sheep Connexin 49 AAF01367 human Connexin 43 P17302Mesocricetus Connexin 50 AAS83438 Cercopithecus Connexin 43 AAR33082Cricetulus Connexin 50 AAR98618 Oryctolagus Connexin 43 AAR33084 ChickConnexin 50 BAA05381 Spermophilus Connexin 43 AAR33086 human Connexin 59AAG09406 Cricetulus Connexin 43 AAO61858

Thus, the provided polypeptide can comprise the amino acid sequence SEQID NO:1, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:90 or ID NO:91 or conservative variants or fragments thereof.

The 20-30 carboxy-terminal-most amino acid sequence of alpha Connexinsare characterized by a distinctive and conserved organization. Thisdistinctive and conserved organization would include a type II PDZbinding motif (Φ-x-Φ; wherein x=any amino acid and Φ=a Hydrophobic aminoacid; e.g., Table 2, BOLD) and proximal to this motif, Proline (P)and/or Glycine (G) hinge residues; a high frequency phospho-Serine (S)and/or phospho-Threonine (T) residues; and a high frequency ofpositively charged Arginine (R), Lysine (K) and negatively chargedAspartic acid (D) or Glutamic acid (E) amino acids. For many alphaConnexins, the P and G residues occur in clustered motifs (e.g., Table2, italicized) proximal to the carboxy-terminal type II PDZ bindingmotif. The S and T phosphor-amino acids of most alpha Connexins also aretypically organized in clustered, repeat-like motifs (e.g., Table 2,underlined). This organization is particularly the case for Cx43, where90% of 20 carboxyl terminal-most amino acids are comprised of the latterseven amino acids. In a further example of the high conservation of thesequence, ACT peptide organization of Cx43 is highly conserved fromhumans to fish (e.g., compare Cx43 ACT sequences for humans andzebrafish in Table 2). In another example, the ACT peptide organizationof Cx45 is highly conserved from humans to birds (e.g., compare Cx45 ACTsequences for humans and chick in Table 2).). In another example, theACT peptide organization of Cx36 is highly conserved from primates tofish (e.g., compare Cx36 ACT sequences for chimp and zebrafish in Table2).

TABLE 2  Alpha Connexin Carboxy-Terminal (ACT) Amino Acid Sequences GeneSequence SEQ ID NO Human alpha Cx43 P SSRA SSRA SSR PRP D DLEI(SEQ ID NO: 1) Chick alpha Cx43 P S RA SSRA SSR PRP D DLEI(SEQ ID NO: 29) Zebrafish alpha P CSRA SSRM SSRA R P D DLDV(SEQ ID NO: 90) Cx43 Human alpha Cx45 G SNKS TA SSKS GDG KN SVWI(SEQ ID NO: 30) Chick alpha Cx45 G SNKSS A SSKS GDG KN SVWI(SEQ ID NO: 31) Human alpha Cx46 G RA SKAS RASS 

RAR

 E DLAI SEQ ID NO: 32) Human alpha Cx46.6 G SASS RD 

 K TVWI (SEQ ID NO: 33) Chimp alpha Cx36 P RVSV PNFG R TQ SSD S AYV(SEQ ID NO: 34) Chick alpha Cx36 P RMSM PNFG R TQ SSD S  AYV(SEQ ID NO: 35) Zebrafish alpha P RMSM PNFG R TQ SSD S  AYV(SEQ ID NO: 91) Cx36 Human alpha Cx47 P RAGSEK G SASS R DG KT TVWI(SEQ LD NO: 36) Human alpha Cx40 G HRL 

H

 YHSDKRRL SKASS KARSD DLSV (SEQ ID NO: 37) Human alpha Cx50P ELTTDDAR P LSRL SKASS RARSD DLTV (SEQ ID NO: 38) Human alpha Cx59P NHVV SLTN NLI GRRVP T DLQI (SEQ ID NO: 39) Rat alpha Cx33P S CV SSS A VLTTIC SS DQVV PVG L  (SEQ ID NO: 40) SS  FYMSheep alpha Cx44 G R SSKA SKSS GG RARAA DLAI (SEQ ID NO: 41)Human beta Cx26 LC YLLIR YCSGK SKKPV (SEQ ID NO: 42)

Thus, in one aspect, the provided polypeptide comprises one, two, threeor all of the amino acid motifs selected from the group consisting of 1)a type II PDZ binding motif, 2) Proline (P) and/or Glycine (G) hingeresidues; 3) clusters of phospho-Serine (S) and/or phospho-Threonine (T)residues; and 4) a high frequency of positively charged Arginine (R) andLysine (K) and negatively charged Aspartic acid (D) and/or Glutamic acid(E) amino acids). In another aspect, the provided polypeptide comprisesa type II PDZ binding motif at the carboxy-terminus, Proline (P) and/orGlycine (G) hinge residues proximal to the PDZ binding motif, andpositively charged residues (K, R, D, E) proximal to the hinge residues.

PDZ domains were originally identified as conserved sequence elementswithin the postsynaptic density protein PSD95/SAP90, the Drosophilatumor suppressor dlg-A, and the tight junction protein ZO-1. Althoughoriginally referred to as GLGF or DHR motifs, they are now known by anacronym representing these first three PDZ-containing proteins(PSD95/DLG/ZO-1). These 80-90 amino acid sequences have now beenidentified in well over 75 proteins and are characteristically expressedin multiple copies within a single protein. Thus, in one aspect, theprovided polypeptide can inhibit the binding of an alpha Connexin to aprotein comprising a PDZ domain. The PDZ domain is a specific type ofprotein-interaction module that has a structurally well-definedinteraction ‘pocket’ that can be filled by a PDZ-binding motif, referredto herein as a “PDZ motif”. PDZ motifs are consensus sequences that arenormally, but not always, located at the extreme intracellular carboxylterminus. Four types of PDZ motifs have been classified: type I(S/T-x-Φ), type II (Φ-x-Φ), type III (Ψ-x-Φ) and type IV (D-x-V), wherex is any amino acid, Φ is a hydrophobic residue (V, I, L, A, G, W, C, M,F) and Ψ is a basic, hydrophilic residue (H, R, K). (Songyang, Z., etal. 1997. Science 275, 73-77). Thus, in one aspect, the providedpolypeptide comprises a type II PDZ binding motif.

It is noted that the 18 carboxy-terminal-most amino acid sequence ofalpha Cx37 represents an exceptional variation on the ACT peptide theme.The Cx37 ACT-like sequence is GQKPPSRPSSSASKKQ*YV (SEQ ID NO: 43). Thusthe carboxy terminal 4 amino acids of Cx37 conform only in part to atype II PDZ binding domain. Instead of a classical type II PDZ bindingdomain, Cx37 has a neutral Q* at position 2 where a hydrophobic aminoacid would be expected. As such Cx37 comprises what might be termed atype II PDZ binding domain-like sequence. Nonetheless, Cx37 strictlymaintains all other aspects of ACT peptide organization includingclustered serine residues, frequent R and K residues and a P-richsequence proximal to the PDZ binding domain-like sequence. Given thisoverall level of conservation of ACT-like organization in common withthe other >70 alpha Connexins listed above, it is understood that theCx37 ACT-like carboxy terminus functions in the provided capacity.

For comparison, the beta Connexin Cx26 is shown in Table 2. Cx26 has nocarboxyl terminal type II PDZ binding motif; less than 30% of thecarboxyl terminal most amino acids comprise S, T, R, D or E residues; ithas no evidence of motifs proximal to a type II PDZ binding motif or PDZbinding like motif containing clusters of P and G hinge residues; and noevidence of clustered, repeat-like motifs of serine and threoninephospho-amino acids. Cx26 does have three Lysine (K) residues, clusteredone after the other near the carboxy terminus of the sequence. However,no alpha Connexin surveyed in the >70 alpha Connexins listed above wasfound to display this feature of three repeated K residues domain atcarboxy terminus (Cx26 is a beta connexin, thus by definition does nothave an ACT domain).

As provided herein, the unique functional characteristics of thisrelatively short stretch of amino acids encompass unexpected roles inreducing inflammation, promoting healing, reducing scarring, increasingtensile strength, and promoting regeneration of complex tissue structureand function following injury in tissues as diverse as skin and brain.Thus, in one aspect, the provided polypeptide comprises a type II PDZbinding motif (Φ-x-Φ; wherein x=any amino acid and Φ=a Hydrophobic aminoacid). In another aspect, greater than 50%, 60%, 70%, 80%, 90% of theamino acids of the provided ACT polypeptide is comprised one or more ofProline (P), Glycine (G), phospho-Serine (S), phospho-Threonine (T),Arginine (R), Lysine (K), Aspartic acid (D), or Glutamic acid (E) aminoacid residues.

The amino acids Proline (P), Glycine (G), Arginine (R), Lysine (K),Aspartic acid (D), and Glutamic acid (E) are necessary determinants ofprotein structure and function. Proline and Glycine residues provide fortight turns in the 3D structure of proteins, enabling the generation offolded conformations of the polypeptide required for function. Chargedamino acid sequences are often located at the surface of folded proteinsand are necessary for chemical interactions mediated by the polypeptideincluding protein-protein interactions, protein-lipid interactions,enzyme-substrate interactions and protein-nucleic acid interactions.Thus, in another aspect Proline (P) and Glycine (G) Lysine (K), Asparticacid (D), and Glutamic acid (E) rich regions proximal to the type II PDZbinding motif provide for properties necessary to the provided actionsof ACT peptides. In another aspect, the provided polypeptide comprisesProline (P) and Glycine (G) Lysine (K), Aspartic acid (D), and/orGlutamic acid (E) rich regions proximal to the type II PDZ bindingmotif.

Phosphorylation is the most common post-translational modification ofproteins and is crucial for modulating or modifying protein structureand function. Aspects of protein structure and function modified byphosphorylation include protein conformation, protein-proteininteractions, protein-lipid interactions, protein-nucleic acidinteractions, channel gating, protein trafficking and protein turnover.Thus, in one aspect the phospho-Serine (S) and/or phospho-Threonine (T)rich sequences are necessary for modifying the function of ACT peptides,increasing or decreasing efficacy of the polypeptides in their providedactions. In another aspect, the provided polypeptide comprise Serine (S)and/or phospho-Threonine (T) rich sequences or motifs.

In another example, respecting definition of an ACT peptide, it ishighly auspicious, in light of the high degree of tissue/organregeneration potential in lower animals such as fish, that a methionineoccurs near the amino terminus of the ACT sequence of zebrafish Cx43(Table 2). In addition to encoding methionine, the methionine base pairtriplet is an alternate translation start site. If translation initiatedfrom this methionine, the sequence SSRARPDDLDV (SEQ ID NO:90), would beproduced. This translation product maintains all the conserved anddistinctive features of a canonical ACT peptide. Specifically thispeptide comprises a carboxy terminal type II PDZ binding domain and hasa domain enriched in P, R and D residues proximal to the PDZ bindingdomain. In addition, the sequence comprises a clustered S motif, withpotential to modulate ACT peptide function at its amino terminal. Thisraises the interesting prospect that animals with high tissue/organregeneration potential such as fish may translate ACT peptides sequencesdirectly.

Thus, the provided polypeptide can comprise the c-terminal sequence ofhuman Cx43. Thus, the provided polypeptide can comprise the amino acidsequence SEQ ID NO:1 or SEQ ID NO:2. The polypeptide can comprise 9amino acids of the carboxy terminus of human Cx40. Thus, the polypeptidecan comprise the amino acid sequence SEQ ID NO:5.

When specific proteins are referred to herein, variants, derivatives,and fragments are contemplated. Protein variants and derivatives arewell understood to those of skill in the art and in can involve aminoacid sequence modifications. For example, amino acid sequencemodifications typically fall into one or more of three classes:substitutional, insertional or deletional variants. Insertions includeamino and/or carboxyl terminal fusions as well as intrasequenceinsertions of single or multiple amino acid residues. Insertionsordinarily will be smaller insertions than those of amino or carboxylterminal fusions, for example, on the order of one to four residues.Deletions are characterized by the removal of one or more amino acidresidues from the protein sequence. These variants ordinarily areprepared by site specific mutagenesis of nucleotides in the DNA encodingthe protein, thereby producing DNA encoding the variant, and thereafterexpressing the DNA in recombinant cell culture. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known and include, for example, M13 primer mutagenesisand PCR mutagenesis. Amino acid substitutions are typically of singleresidues, but can occur at a number of different locations at once;insertions usually will be on the order of about from 1 to 10 amino acidresidues. Deletions or insertions preferably are made in adjacent pairs,i.e., a deletion of 2 residues or insertion of 2 residues.Substitutions, deletions, insertions or any combination thereof may becombined to arrive at a final construct. The mutations must not placethe sequence out of reading frame and preferably will not createcomplementary regions that could produce secondary mRNA structure unlesssuch a change in secondary structure of the mRNA is desired.Substitutional variants are those in which at least one residue has beenremoved and a different residue inserted in its place. Suchsubstitutions generally are made in accordance with the following Table3 and are referred to as conservative substitutions.

TABLE 3 Amino Acid Substitutions Original Residue ExemplarySubstitutions Ala Ser Arg Lys Asn Gln Asp Glu Cys Ser Gln Asn Glu AspGly Pro His Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln Met Leu; Ile PheMet; Leu; Tyr Pro Gly Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

For example, the replacement of one amino acid residue with another thatis biologically and/or chemically similar is known to those skilled inthe art as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue for another, orone polar residue for another. The substitutions include combinationsshown in Table 3. Conservatively substituted variations of eachexplicitly disclosed sequence are included within the polypeptidesprovided herein.

Typically, conservative substitutions have little to no impact on thebiological activity of a resulting polypeptide. In a particular example,a conservative substitution is an amino acid substitution in a peptidethat does not substantially affect the biological function of thepeptide. A peptide can include one or more amino acid substitutions, forexample 2-10 conservative substitutions, 2-5 conservative substitutions,4-9 conservative substitutions, such as 2, 5 or 10 conservativesubstitutions.

A polypeptide can be produced to contain one or more conservativesubstitutions by manipulating the nucleotide sequence that encodes thatpolypeptide using, for example, standard procedures such assite-directed mutagenesis or PCR. Alternatively, a polypeptide can beproduced to contain one or more conservative substitutions by usingstandard peptide synthesis methods. An alanine scan can be used toidentify which amino acid residues in a protein can tolerate an aminoacid substitution. In one example, the biological activity of theprotein is not decreased by more than 25%, for example not more than20%, for example not more than 10%, when an alanine, or otherconservative amino acid (such as those listed below), is substituted forone or more native amino acids.

Further information about conservative substitutions can be found in,among other locations, in Ben-Bassat et al., (J. Bacterial. 169:751-7,1987), O'Regan et al., (Gene 77:237-51, 1989), Sahin-Toth et al.,(Protein Sci. 3:240-7, 1994), Hochuli et al., (Bio/Technology 6:1321-5,1988) and in standard textbooks of genetics and molecular biology.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also may be desirable.Deletions or substitutions of potential proteolysis sites, e.g. Arg, isaccomplished for example by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and asparyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Otherpost-translational modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the o-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W. H. Freeman & Co., San Francisco pp 79-86[1983]), acetylation of the N-terminal amine and, in some instances,amidation of the C-terminal carboxyl.

It is understood that there are numerous amino acid and peptide analogswhich can be incorporated into the disclosed compositions. For example,there are numerous D amino acids or amino acids which have a differentfunctional substituent than the amino acids shown in Table 3. Theopposite stereoisomers of naturally occurring peptides are disclosed, aswell as the stereoisomers of peptide analogs. These amino acids canreadily be incorporated into polypeptide chains by charging tRNAmolecules with the amino acid of choice and engineering geneticconstructs that utilize, for example, amber codons, to insert the analogamino acid into a peptide chain in a site specific way (Thorson et al.,Methods in Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion inBiotechnology, 3:348-354 (1992); Ibba, Biotechnology & GeneticEngineering Reviews 13:197-216 (1995), Cahill et al., TIBS,14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba andHennecke, Bio/technology, 12:678-682 (1994), all of which are hereinincorporated by reference at least for material related to amino acidanalogs).

Molecules can be produced that resemble polypeptides, but which are notconnected via a natural peptide linkage. For example, linkages for aminoacids or amino acid analogs can include CH₂NH—, —CH₂S—, —CH₂—CH₂—,—CH═CH— (cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CHH₂SO— (These andothers can be found in Spatola, A. F. in Chemistry and Biochemistry ofAmino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker,New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1,Issue 3, Peptide Backbone Modifications (general review); Morley, TrendsPharm Sci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res14:177-185 (1979) (—CH₂NH—, CH₂CH₂—); Spatola et al. Life Sci38:1243-1249 (1986) (—CH H₂—S); Hann J. Chem. Soc Perkin Trans. I307-314 (1982) (—CH—CH—, cis and trans); Almquist et al. J Med. Chem.23:1392-1398 (1980) (—COCH₂—); Jennings-White et al. Tetrahedron Lett23:2533 (1982) (—COCH₂—); Szelke et al. European Appln, EP 45665 CA(1982): 97:39405 (1982) (—CH(OH)CH₂—); Holladay et al. Tetrahedron. Lett24:4401-4404 (1983) (—C(OH)CH₂—); and Hruby Life Sci 31:189-199 (1982)(—CH₂—S—); each of which is incorporated herein by reference. It isunderstood that peptide analogs can have more than one atom between thebond atoms, such as b-alanine, g-aminobutyric acid, and the like.

Amino acid analogs and peptide analogs often have enhanced or desirableproperties, such as, more economical production, greater chemicalstability, enhanced pharmacological properties (half-life, absorption,potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum ofbiological activities), reduced antigenicity, greater ability to crossbiological barriers (e.g., gut, blood vessels, blood-brain-barrier), andothers.

D-amino acids can be used to generate more stable peptides, because Damino acids are not recognized by peptidases and such. Systematicsubstitution of one or more amino acids of a consensus sequence with aD-amino acid of the same type (e.g., D-lysine in place of L-lysine) canbe used to generate more stable peptides. Cysteine residues can be usedto cyclize or attach two or more peptides together. This can bebeneficial to constrain peptides into particular conformations. (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference).

Thus, the provided polypeptide can comprise a conservative variant ofthe c-terminus of an alpha Connexin (ACT). As shown in Table 4, anexample of a single conservative substitution within the sequence SEQ IDNO:2 is given in the sequence SEQ ID NO:3. An example of threeconservative substitutions within the sequence SEQ ID NO:2 is given inthe sequence SEQ ID NO:4. Thus, the provided polypeptide can comprisethe amino acid SEQ ID NO:3 or SEQ ID NO:4.

TABLE 4  ACT Polypeptide Variants Sequence SEQ ID NO RPRPDDLEISEQ ID NO: 2 RPRPDDLEV SEQ ID NO: 3 RPRPDDVPV SEQ ID NO: 4SSRASSRASSRPRPDDLEV SEQ ID NO: 44 RPKPDDLEI SEQ ID NO: 45SSRASSRASSRPKPDDLEI SEQ ID NO: 46 RPKPDDLDI SEQ ID NO: 47SSRASSRASSRPRPDDLDI SEQ ID NO: 48 SSRASTRASSRPRPDDLEI SEQ ID NO: 49RPRPEDLEI SEQ ID NO: 50 SSRASSRASSRPRPEDLEI SEQ ID NO: 51 GDGKNSVWVSEQ ID NO: 52 SKAGSNKSTASSKSGDGKNSVWV SEQ ID NO: 53 GQKPPSRPSSSASKKLYVSEQ ID NO: 54

It is understood that one way to define any variants, modifications, orderivatives of the disclosed genes and proteins herein is throughdefining the variants, modification, and derivatives in terms ofsequence identity (also referred to herein as homology) to specificknown sequences. Specifically disclosed are variants of the nucleicacids and polypeptides herein disclosed which have at least 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent sequenceidentity to the stated or known sequence. Those of skill in the artreadily understand how to determine the sequence identity of twoproteins or nucleic acids. For example, the sequence identity can becalculated after aligning the two sequences so that the sequenceidentity is at its highest level.

Another way of calculating sequence identity can be performed bypublished algorithms. Optimal alignment of sequences for comparison maybe conducted by the local sequence identity algorithm of Smith andWaterman Adv. Appl. Math. 2: 482 (1981), by the sequence identityalignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443(1970), by the search for similarity method of Pearson and Lipman, Proc.Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementationsof these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by inspection. These references are incorporatedherein by reference in their entirety for the methods of calculatingsequence identity.

The same types of sequence identity can be obtained for nucleic acidsby, for example, the algorithms disclosed in Zuker, M. Science244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710,1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989 which are hereinincorporated by reference for at least material related to nucleic acidalignment.

Thus, the provided polypeptide can comprise an amino acid sequence withat least 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 percent sequence identity to the c-terminus of an alpha Connexin(ACT). Thus, in one aspect, the provided polypeptide comprises an aminoacid sequence with at least 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99 percent sequence identity to SEQ ID NO:1, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO: 90 or SEQ ID NO:91. As anexample, provided is a polypeptide (SEQ ID NO:4) having 66% sequenceidentity to the same stretch of 9 amino acids occurring on thecarboxy-terminus of human Cx43 (SEQ ID NO:2).

The herein provided polypeptides can be added directly to a tissueinjury in a subject. However, efficiency of cytoplasmic localization ofthe provided polypeptide is enhanced by cellular internalizationtransporter chemically linked in cis or trans with the polypeptide.Efficiency of cell internalization transporters are enhanced further bylight or co-transduction of cells with Tat-HA peptide.

Thus, the provided polypeptide can comprise a cellular internalizationtransporter or sequence. The cellular internalization sequence can beany internalization sequence known or newly discovered in the art, orconservative variants thereof. Non-limiting examples of cellularinternalization transporters and sequences include Antennapediasequences, TAT, HIV-Tat, Penetratin, Antp-3A (Antp mutant), Buforin II,Transportan, MAP (model amphipathic peptide), K-FGF, Ku70, Prion, pVEC,Pep-1, SynB1, Pep-7, HN-1, BGSC (Bis-Guanidinium-Spermidine-Cholesterol,and BGTC (Bis-Guanidinium-Tren-Cholesterol) (see Table 5).

TABLE 5 Cell Internalization Transporters Name Sequence SEQ ID NO AntpRQPKIWFPNRRKPWKK (SEQ ID NO: 7) HIV-Tat GRKKRRQRPPQ (SEQ ID NO: 14)Penetratin RQIKIWFQNRRMKWKK (SEQ ID NO: 15) Antp-3A RQIAIWFQNRRMKWAA(SEQ ID NO: 16) Tat RKKRRQRRR (SEQ ID NO: 17) Buforin IITRSSRAGLQFPVGRVHRLLRK (SEQ ID NO: 18) Transportan GWTLNSAGYLLGKINKALAALA(SEQ ID NO: 19) KKIL model amphipathic  KLALKLALKALKAALKLA(SEQ ID NO: 20) peptide (MAP) K-FGF AAVALLPAVLLALLAP (SEQ ID NO: 21)Ku70 VPMLK-PMLKE (SEQ ID NO: 22) Prion MANLGYWLLALFVTMWTDVGL(SEQ ID NO: 23) CKKRPKP pVEC LLIILRRRIRKQAHAHSK (SEQ ID NO: 24) Pep-1KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 25) SynB1 RGGRLSYSRRRFSTSTGR(SEQ ID NO: 26) Pep-7 SDLWEMMMVSLACQY (SEQ ID NO: 27) HN-1 TSPLNIHNGQKL(SEQ ID NO: 28) BGSC (Bis- Guanidinium- Spermidine- Cholesterol)

BGTC (Bis- Guanidinium-Tren- Cholesterol)

Thus, the provided polypeptide can further comprise the amino acidsequence SEQ ID NO:7, SEQ ID NO:14 (Bucci, M. et al. 2000. Nat. Med. 6,1362-1367), SEQ ID NO:15 (Derossi, D., et al. 1994. Biol. Chem. 269,10444-10450), SEQ ID NO:16 (Fischer, P. M. et al. 2000. J. Pept. Res.55, 163-172), SEQ ID NO:17 (Frankel, A. D. & Pabo, C. O. 1988. Cell 55,1189-1193; Green, M. & Loewenstein, P. M. 1988. Cell 55, 1179-1188), SEQID NO:18 (Park, C. B., et al. 2000. Proc. Natl Acad. Sci. USA 97,8245-8250), SEQ ID NO:19 (Pooga, M., et al. 1998. FASEB J. 12, 67-77),SEQ ID NO:20 (Oehlke, J. et al. 1998. Biochim. Biophys. Acta. 1414,127-139), SEQ ID NO:21 (Lin, Y. Z., et al. 1995. J. Biol. Chem. 270,14255-14258), SEQ ID NO:22 (Sawada, M., et al. 2003. Nature Cell Biol.5, 352-357), SEQ ID NO:23 (Lundberg, P. et al. 2002. Biochem. Biophys.Res. Commun. 299, 85-90), SEQ ID NO:24 (Elmquist, A., et al. 2001. Exp.Cell Res. 269, 237-244), SEQ ID NO:25 (Morris, M. C., et al. 2001.Nature Biotechnol. 19, 1173-1176), SEQ ID NO:26 (Rousselle, C. et al.2000. Mol. Pharmacol. 57, 679-686), SEQ ID NO:27 (Gao, C. et al. 2002.Bioorg. Med. Chem. 10, 4057-4065), or SEQ ID NO:28 (Hong, F. D. &Clayman, G. L. 2000. Cancer Res. 60, 6551-6556). The providedpolypeptide can further comprise BGSC(Bis-Guanidinium-Spermidine-Cholesterol) or BGTC(Bis-Guanidinium-Tren-Cholesterol) (Vigneron, J. P. et al. 1998. Proc.Natl. Acad. Sci. USA. 93, 9682-9686). The preceding references arehereby incorporated herein by reference in their entirety for theteachings of cellular internalization vectors and sequences. Any otherinternalization sequences now known or later identified can be combinedwith a peptide of the invention.

The provided polypeptide can comprise any ACT sequence (e.g, any of theACT peptides disclosed herein) in combination with any of the hereinprovided cell internalization sequences. Examples of said combinationsare given in Table 6. Thus, the provided polypeptide can comprise anAntennapedia sequence comprising amino acid sequence SEQ ID NO:7. Thus,the provided polypeptide can comprise the amino acid sequence SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

TABLE 6  ACT Polypeptides with Cell Internalization Sequences (CIS)CIS/ACT Sequence SEQ ID NO Antp/ACT 2 RQPKIWFPNRRKPWKK  SEQ ID NO: 8PSSRASSRASSRPRPDDLEI Antp/ACT 1 RQPKIWFPNRRKPWKK  SEQ ID NO: 9 RPRPDDLEIAntp/ACT 3 RQPKIWFPNRRKPWKK  SEQ ID NO: 10 RPRPDDLEV Antp/ACT 4RQPKIWFPNRRKPWKK  SEQ ID NO: 11 RPRPDDVPV Antp/ACT 5 RQPKIWFPNRRKPWKK SEQ ID NO: 12 KARSDDLSV HIV-Tat/ GRKKRRQRPPQ RPRPDDLEI SEQ ID NO: 56ACT 1 Penetratin/ RQIKIWFQNRRMKWKK  SEQ ID NO: 57 ACT 1 RPRPDDLEIAntp-3A/ RQIAIWFQNRRMKWAA  SEQ ID NO: 58 ACT 1 RPRPDDLEI Tat/ACT 1RKKRRQRRR RPRPDDLEI SEQ ID NO: 59 Buforin II/ TRSSRAGLQFPVGRVHRLLRK SEQ ID NO: 60 ACT 1 RPRPDDLEI Transportan/ GWTLNSAGYLLGKINKALAALSEQ ID NO: 61 ACT 1 AKKIL RPRPDDLEI MAP/ACT 1 KLALKLALKALKAALKLA SEQ ID NO: 62 RPRPDDLEI K-FGF/ACT 1 AAVALLPAVLLALLAP  SEQ ID NO: 63RPRPDDLEI Ku70/ACT 1 VPMLKPMLKE RPRPDDLEI SEQ ID NO: 64 Prion/ACT 1MANLGYWLLALFVTMWTDVG SEQ ID NO: 65 LCKKRPKP RPRPDDLEI pVEC/ACT 1LLIILRRRIRKQAHAHSK  SEQ ID NO: 66 RPRPDDLEI Pep-1/ACT 1KETWWETWWTEWSQPKKKRKV  SEQ ID NO: 67 RPRPDDLEI SynB1/ACT 1RGGRLSYSRRRFSTSTGR  SEQ ID NO: 68 RPRPDDLEI Pep-7/ACT 1 SDLWEMMMVSLACQY SEQ ID NO: 69 RPRPDDLEI HN-1/ACT 1 TSPLNIHNGQKL RPRPDDLEI SEQ ID NO: 70

Also provided are isolated nucleic acids encoding the polypeptidesprovided herein. The disclosed nucleic acids are made up of for example,nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limitingexamples of these and other molecules are discussed herein. It isunderstood that for example, when a vector is expressed in a cell, theexpressed mRNA will typically be made up of A, C, G, and U.

By “isolated nucleic acid” or “purified nucleic acid” is meant DNA thatis free of the genes that, in the naturally-occurring genome of theorganism from which the DNA of the invention is derived, flank the gene.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector, such as an autonomously replicating plasmidor virus; or incorporated into the genomic DNA of a prokaryote oreukaryote (e.g., a transgene); or which exists as a separate molecule(e.g., a cDNA or a genomic or cDNA fragment produced by PCR, restrictionendonuclease digestion, or chemical or in vitro synthesis). It alsoincludes a recombinant DNA which is part of a hybrid gene encodingadditional polypeptide sequence. The term “isolated nucleic acid” alsorefers to RNA, e.g., an mRNA molecule that is encoded by an isolated DNAmolecule, or that is chemically synthesized, or that is separated orsubstantially free from at least some cellular components, e.g., othertypes of RNA molecules or polypeptide molecules.

Thus, provided is an isolated nucleic acid encoding a polypeptidecomprising the amino acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

Thus, the provided nucleic acid can comprise the nucleic acid sequenceSEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83,SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87 SEQ ID NO:88, orSEQ ID NO:89.

The herein provided nucleic acid can be operably linked to an expressioncontrol sequence. Also provided is a vector comprising one or more ofthe herein provided nucleic acids, wherein the nucleic acid is operablylinked to an expression control sequence. There are a number ofcompositions and methods which can be used to deliver nucleic acids tocells, either in vitro or in vivo. These methods and compositions canlargely be broken down into two classes: viral based delivery systemsand non-viral based delivery systems. For example, the nucleic acids canbe delivered through a number of direct delivery systems such as,electroporation, lipofection, calcium phosphate precipitation, plasmids,viral vectors, viral nucleic acids, phage nucleic acids, phages,cosmids, or via transfer of genetic material in cells or carriers suchas cationic liposomes. Appropriate means for transfection, includingviral vectors, chemical transfectants, or physico-mechanical methodssuch as electroporation and direct diffusion of DNA, are described by,for example, Wolff, J. A., et al., Science, 247, 1465-1468, (1990); andWolff, J. A. Nature, 352, 815-818, (1991). Such methods are well knownin the art and readily adaptable for use with the compositions andmethods described herein. In certain cases, the methods will be modifiedto specifically function with large DNA molecules. Further, thesemethods can be used to target certain diseases and cell populations byusing the targeting characteristics of the carrier.

Transfer vectors can be any nucleotide construction used to delivergenes into cells (e.g., a plasmid), or as part of a general strategy todeliver genes, e.g., as part of recombinant retrovirus or adenovirus(Ram et al. Cancer Res. 53:83-88, (1993)).

As used herein, plasmid or viral vectors are agents that transport thedisclosed nucleic acids, such as SEQ ID NO:6, into the cell withoutdegradation and include a promoter yielding expression of the gene inthe cells into which it is delivered. In some embodiments the promotersare derived from either a virus or a retrovirus. Viral vectors are, forexample, Adenovirus, Adeno-associated virus, Herpes virus, Vacciniavirus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis andother RNA viruses, including these viruses with the HIV backbone. Alsodisclosed are any viral families which share the properties of theseviruses which make them suitable for use as vectors. Retrovirusesinclude Murine Maloney Leukemia virus, MMLV, and retroviruses thatexpress the desirable properties of MMLV as a vector. Retroviral vectorsare able to carry a larger genetic payload, i.e., a transgene or markergene, than other viral vectors, and for this reason are a commonly usedvector. However, they are not as useful in non-proliferating cells.Adenovirus vectors are relatively stable and easy to work with, havehigh titers, and can be delivered in aerosol formulation, and cantransfect non-dividing cells. Pox viral vectors are large and haveseveral sites for inserting genes, they are thermostable and can bestored at room temperature. Also disclosed is a viral vector which hasbeen engineered so as to suppress the immune response of the hostorganism, elicited by the viral antigens. Vectors of this type can carrycoding regions for Interleukin 8 or 10.

Viral vectors can have higher transaction (ability to introduce genes)abilities than chemical or physical methods to introduce genes intocells. Typically, viral vectors contain, nonstructural early genes,structural late genes, an RNA polymerase III transcript, invertedterminal repeats necessary for replication and encapsidation, andpromoters to control the transcription and replication of the viralgenome. When engineered as vectors, viruses typically have one or moreof the early genes removed and a gene or gene/promoter cassette isinserted into the viral genome in place of the removed viral DNA.Constructs of this type can carry up to about 8 kb of foreign geneticmaterial. The necessary functions of the removed early genes aretypically supplied by cell lines which have been engineered to expressthe gene products of the early genes in trans.

A retrovirus is an animal virus belonging to the virus family ofRetroviridae, including any types, subfamilies, genus, or tropisms.Retroviral vectors, in general, are described by Verma, I. M.,Retroviral vectors for gene transfer. In Microbiology-1985, AmericanSociety for Microbiology, pp. 229-232, Washington, (1985), which isincorporated by reference herein. Examples of methods for usingretroviral vectors for gene therapy are described in U.S. Pat. Nos.4,868,116 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136;and Mulligan, (Science 260:926-932 (1993)); the teachings of which areincorporated herein by reference.

A retrovirus is essentially a package which has packed into it nucleicacid cargo. The nucleic acid cargo carries with it a packaging signal,which ensures that the replicated daughter molecules will be efficientlypackaged within the package coat. In addition to the package signal,there are a number of molecules which are needed in cis, for thereplication, and packaging of the replicated virus. Typically aretroviral genome, contains the gag, pol, and env genes which areinvolved in the making of the protein coat. It is the gag, pol, and envgenes which are typically replaced by the foreign DNA that it is to betransferred to the target cell. Retrovirus vectors typically contain apackaging signal for incorporation into the package coat, a sequencewhich signals the start of the gag transcription unit, elementsnecessary for reverse transcription, including a primer binding site tobind the tRNA primer of reverse transcription, terminal repeat sequencesthat guide the switch of RNA strands during DNA synthesis, a purine richsequence 5′ to the 3′ LTR that serve as the priming site for thesynthesis of the second strand of DNA synthesis, and specific sequencesnear the ends of the LTRs that enable the insertion of the DNA state ofthe retrovirus to insert into the host genome. The removal of the gag,pol, and env genes allows for about 8 kb of foreign sequence to beinserted into the viral genome, become reverse transcribed, and uponreplication be packaged into a new retroviral particle. This amount ofnucleic acid is sufficient for the delivery of a one to many genesdepending on the size of each transcript.

Since the replication machinery and packaging proteins in mostretroviral vectors have been removed (gag, pol, and env), the vectorsare typically generated by placing them into a packaging cell line. Apackaging cell line is a cell line which has been transfected ortransformed with a retrovirus that contains the replication andpackaging machinery, but lacks any packaging signal. When the vectorcarrying the DNA of choice is transfected into these cell lines, thevector containing the gene of interest is replicated and packaged intonew retroviral particles, by the machinery provided in cis by the helpercell. The genomes for the machinery are not packaged because they lackthe necessary signals.

The construction of replication-defective adenoviruses has beendescribed (Berkner et al., J. Virology 61:1213-1220 (1987); Massie etal., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., J. Virology57:267-274 (1986); Davidson et al., J. Virology 61:1226-1239 (1987);Zhang “Generation and identification of recombinant adenovirus byliposome-mediated transfection and PCR analysis” BioTechniques15:868-872 (1993)). The benefit of the use of these viruses as vectorsis that they are limited in the extent to which they can spread to othercell types, since they can replicate within an initial infected cell,but are unable to form new infectious viral particles. Recombinantadenoviruses have been shown to achieve high efficiency gene transferafter direct, in vivo delivery to airway epithelium, hepatocytes,vascular endothelium, CNS parenchyma and a number of other tissue sites(Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin.Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092(1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992);Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout,Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993);Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen.Virology 74:501-507 (1993)). Recombinant adenoviruses achieve genetransduction by binding to specific cell surface receptors, after whichthe virus is internalized by receptor-mediated endocytosis, in the samemanner as wild type or replication-defective adenovirus (Chardonnet andDales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985);Seth, et al., J. Virol. 51:650-655 (1984); Seth, et al., Mol. Cell.Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991);Wickham et al., Cell 73:309-319 (1993)).

A viral vector can be one based on an adenovirus which has had the E1gene removed, and these virons are generated in a cell line such as thehuman 293 cell line. In one aspect, both the E1 and E3 genes are removedfrom the adenovirus genome. Another type of viral vector is based on anadeno-associated virus (AAV). This defective parvovirus can infect manycell types and is nonpathogenic to humans. AAV type vectors cantransport about 4 to 5 kb and wild type AAV is known to stably insertinto chromosome 19. As an example, this vector can be the P4.1 C vectorproduced by Avigen, San Francisco, Calif., which can contain the herpessimplex virus thymidine kinase gene, HSV-tk, and/or a marker gene, suchas the gene encoding the green fluorescent protein, GFP.

In another type of AAV virus, the AAV contains a pair of invertedterminal repeats (ITRs) which flank at least one cassette containing apromoter which directs cell-specific expression operably linked to aheterologous gene. Heterologous in this context refers to any nucleotidesequence or gene which is not native to the AAV or B19 parvovirus.

Typically the AAV and B19 coding regions have been deleted, resulting ina safe, noncytotoxic vector. The AAV ITRs, or modifications thereof,confer infectivity and site-specific integration, but not cytotoxicity,and the promoter directs cell-specific expression. U.S. Pat. No.6,261,834 is herein incorporated by reference for material related tothe AAV vector.

The disclosed vectors thus provide DNA molecules which are capable ofintegration into a mammalian chromosome without substantial toxicity.

The inserted genes in viral and retroviral usually contain promoters,and/or enhancers to help control the expression of the desired geneproduct. A promoter is generally a sequence or sequences of DNA thatfunction when in a relatively fixed location in regard to thetranscription start site. A promoter contains core elements required forbasic interaction of RNA polymerase and transcription factors, and maycontain upstream elements and response elements.

Molecular genetic experiments with large human herpes viruses haveprovided a means whereby large heterologous DNA fragments can be cloned,propagated and established in cells permissive for infection with herpesviruses (Sun et al., Nature genetics 8: 33-41, 1994; Cotter andRobertson, Curr Opin Mol Ther 5: 633-644, 1999). These large DNA viruses(herpes simplex virus (HSV) and Epstein-Barr virus (EBV), have thepotential to deliver fragments of human heterologous DNA >150 kb tospecific cells. EBV recombinants can maintain large pieces of DNA in theinfected B-cells as episomal DNA. Individual clones carried humangenomic inserts up to 330 kb appeared genetically stable. Themaintenance of these episomes requires a specific EBV nuclear protein,EBNA1, constitutively expressed during infection with EBV. Additionally,these vectors can be used for transfection, where large amounts ofprotein can be generated transiently in vitro. Herpesvirus ampliconsystems are also being used to package pieces of DNA >220 kb and toinfect cells that can stably maintain DNA as episomes.

Other useful systems include, for example, replicating andhost-restricted non-replicating vaccinia virus vectors.

The disclosed compositions can be delivered to the target cells in avariety of ways. For example, the compositions can be delivered throughelectroporation, or through lipofection, or through calcium phosphateprecipitation. The delivery mechanism chosen will depend in part on thetype of cell targeted and whether the delivery is occurring for examplein vivo or in vitro.

Thus, the compositions can comprise, in addition to the disclosedpolypeptides, nucleic acids or vectors, for example, lipids such asliposomes, such as cationic liposomes (e.g., DOTMA, DOPE,DC-cholesterol) or anionic liposomes. Liposomes can further compriseproteins to facilitate targeting a particular cell, if desired.Administration of a composition comprising a compound and a cationicliposome can be administered to the blood afferent to a target organ orinhaled into the respiratory tract to target cells of the respiratorytract. Regarding liposomes, see, e.g., Brigham et al. Am. J. Resp. Cell.Mol. Biol. 1:95-100 (1989); Felgner et al. Proc. Natl. Acad. Sci USA84:7413-7417 (1987); U.S. Pat. No. 4,897,355. Furthermore, the compoundcan be administered as a component of a microcapsule that can betargeted to specific cell types, such as macrophages, or where thediffusion of the compound or delivery of the compound from themicrocapsule is designed for a specific rate or dosage.

In the methods described above which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), delivery of the compositions to cells canbe via a variety of mechanisms. As one example, delivery can be via aliposome, using commercially available liposome preparations such asLIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison, Wis.), as well as other liposomes developedaccording to procedures standard in the art. In addition, the disclosednucleic acid or vector can be delivered in vivo by electroporation, thetechnology for which is available from Genetronics, Inc. (San Diego,Calif.) as well as by means of a SONOPORATION machine (ImaRxPharmaceutical Corp., Tucson, Ariz.).

Nucleic acids that are delivered to cells which are to be integratedinto the host cell genome, typically contain integration sequences.These sequences are often viral related sequences, particularly whenviral based systems are used. These viral integration systems can alsobe incorporated into nucleic acids which are to be delivered using anon-nucleic acid based system of deliver, such as a liposome, so thatthe nucleic acid contained in the delivery system can be come integratedinto the host genome.

Other general techniques for integration into the host genome include,for example, systems designed to promote homologous recombination withthe host genome. These systems typically rely on sequence flanking thenucleic acid to be expressed that has enough homology with a targetsequence within the host cell genome that recombination between thevector nucleic acid and the target nucleic acid takes place, causing thedelivered nucleic acid to be integrated into the host genome. Thesesystems and the methods necessary to promote homologous recombinationare known to those of skill in the art.

The compositions can be delivered to the subject's cells in vivo and/orex vivo by a variety of mechanisms well known in the art (e.g., uptakeof naked DNA, liposome fusion, intramuscular injection of DNA via a genegun, endocytosis and the like).

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The compositions can be introduced into the cells via anygene transfer mechanism, such as, for example, calcium phosphatemediated gene delivery, electroporation, microinjection orproteoliposomes. The transduced cells can then be infused (e.g., in apharmaceutically acceptable carrier) or homotopically transplanted backinto the subject per standard methods for the cell or tissue type.Standard methods are known for transplantation or infusion of variouscells into a subject.

The nucleic acids that are delivered to cells typically containexpression controlling systems. For example, the inserted genes in viraland retroviral systems usually contain promoters, and/or enhancers tohelp control the expression of the desired gene product. A promoter isgenerally a sequence or sequences of DNA that function when in arelatively fixed location in regard to the transcription start site. Apromoter contains core elements required for basic interaction of RNApolymerase and transcription factors, and may contain upstream elementsand response elements.

Promoters controlling transcription from vectors in mammalian host cellsmay be obtained from various sources, for example, the genomes ofviruses such as: polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis-B virus, cytomegalovirus, or from heterologousmammalian promoters, e.g. beta actin promoter. The early and latepromoters of the SV40 virus are conveniently obtained as an SV40restriction fragment which also contains the SV40 viral origin ofreplication (Fiers et al., Nature, 273: 113 (1978)). The immediate earlypromoter of the human cytomegalovirus is conveniently obtained as aHindIII E restriction fragment (Greenway, P. J. et al., Gene 18: 355-360(1982)). Of course, promoters from the host cell or related species alsoare useful herein.

Enhancer generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′(Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3′(Lusky, M. L., et al., Mol. Cell Bio. 3: 1108 (1983)) to thetranscription unit. Furthermore, enhancers can be within an intron(Banerji, J. L. et al., Cell 33: 729 (1983)) as well as within thecoding sequence itself (Osborne, T. F., et al., Mol. Cell Bio. 4: 1293(1984)). They are usually between 10 and 300 bp in length, and theyfunction in cis. Enhancers function to increase transcription fromnearby promoters. Enhancers also often contain response elements thatmediate the regulation of transcription. Promoters can also containresponse elements that mediate the regulation of transcription.Enhancers often determine the regulation of expression of a gene. Whilemany enhancer sequences are now known from mammalian genes (globin,elastase, albumin, α-fetoprotein and insulin), typically one will use anenhancer from a eukaryotic cell virus for general expression. Examplesare the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers.

The promoter and/or enhancer may be specifically activated either bylight or specific chemical events which trigger their function. Systemscan be regulated by reagents such as tetracycline and dexamethasone.There are also ways to enhance viral vector gene expression by exposureto irradiation, such as gamma irradiation, or alkylating chemotherapydrugs.

In certain embodiments the promoter and/or enhancer region can act as aconstitutive promoter and/or enhancer to maximize expression of theregion of the transcription unit to be transcribed. In certainconstructs the promoter and/or enhancer region be active in alleukaryotic cell types, even if it is only expressed in a particular typeof cell at a particular time. A promoter of this type is the CMVpromoter (650 bases). Other such promoters are SV40 promoters,cytomegalovirus (full length promoter), and retroviral vector LTR.

It has been shown that all specific regulatory elements can be clonedand used to construct expression vectors that are selectively expressedin specific cell types such as melanoma cells. The glial fibrillaryacetic protein (GFAP) promoter has been used to selectively expressgenes in cells of glial origin.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) may also contain sequencesnecessary for the termination of transcription which may affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites.The transcription unit can also contain a polyadenylation region. Onebenefit of this region is that it increases the likelihood that thetranscribed unit will be processed and transported like mRNA. Theidentification and use of polyadenylation signals in expressionconstructs is well established. Homologous polyadenylation signals canbe used in the transgene constructs. In certain transcription units, thepolyadenylation region is derived from the SV40 early polyadenylationsignal and consists of about 400 bases. Transcribed units an containother standard sequences alone or in combination with the abovesequences improve expression from, or stability of, the construct.

The viral vectors can include nucleic acid sequence encoding a markerproduct. This marker product is used to determine if the gene has beendelivered to the cell and once delivered is being expressed. Examplemarker genes are the E. Coli lacZ gene, which encodes β-galactosidase,and green fluorescent protein.

In some embodiments the marker may be a selectable marker. Examples ofsuitable selectable markers for mammalian cells are dihydrofolatereductase (DHFR), thymidine kinase, neomycin, neomycin analog G418,hydromycin, and puromycin. When such selectable markers are successfullytransferred into a mammalian host cell, the transformed mammalian hostcell can survive if placed under selective pressure. There are twowidely used distinct categories of selective regimes. The first categoryis based on a cell's metabolism and the use of a mutant cell line whichlacks the ability to grow independent of a supplemented media. Twoexamples are: Chinese hamster ovary (CHO) DHFR-cells and mouseLTK-cells. These cells lack the ability to grow without the addition ofsuch nutrients as thymidine or hypoxanthine Because these cells lackcertain genes necessary for a complete nucleotide synthesis pathway,they cannot survive unless the missing nucleotides are provided in asupplemented media. An alternative to supplementing the media is tointroduce an intact DHFR or TK gene into cells lacking the respectivegenes, thus altering their growth requirements. Individual cells whichwere not transformed with the DHFR or TK gene will not be capable ofsurvival in non-supplemented media.

The second category is dominant selection which refers to a selectionscheme used in any cell type and does not require the use of a mutantcell line. These schemes typically use a drug to arrest growth of a hostcell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, (Southern P. and Berg,P., J. Molec. Appl. Genet. 1:327 (1982)), mycophenolic acid, (Mulligan,R. C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B.et al., Mol. Cell. Biol. 5: 410-413 (1985)). The three examples employbacterial genes under eukaryotic control to convey resistance to theappropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid)or hygromycin, respectively. Others include the neomycin analog G418 andpuramycin.

Also provided is a cell comprising one or more of the herein providedvectors. As used herein, “cell”, “cell line”, and “cell culture” may beused interchangeably and all such designations include progeny. Thedisclosed cell can be any cell used to clone or propagate the vectorsprovided herein. Thus, the cell can be from any primary cell culture orestablished cell line. The method may be applied to any cell, includingprokaryotic or eukaryotic, such as bacterial, plant, animal, and thelike. The cell type can be selected by one skilled in the art based onthe choice of vector and desired use.

Disclosed are animals produced by the process of transfecting a cellwithin the animal with any of the nucleic acid molecules or vectorsdisclosed herein. Disclosed are animals produced by the process oftransfecting a cell within the animal any of the nucleic acid moleculesor vectors disclosed herein, wherein the animal is a mammal. Alsodisclosed are animals produced by the process of transfecting a cellwithin the animal any of the nucleic acid molecules or vectors disclosedherein, wherein the mammal is mouse, rat, rabbit, cow, sheep, pig, orprimate.

Provided is a composition comprising one or more of the herein providedpolypeptides, nucleic acids, or vectors in a pharmaceutically acceptablecarrier. Thus, provided is a composition comprising a combination of twoor more of any of the herein provided ACT polypeptides in apharmaceutically acceptable carrier. For example, provided is acomposition comprising SEQ ID NO:1 and SEQ ID NO:5 in a pharmaceuticallyacceptable carrier.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to a subject, along with the nucleic acid or vector,without causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained. The carrier wouldnaturally be selected to minimize any degradation of the activeingredient and to minimize any adverse side effects in the subject, aswould be well known to one of skill in the art.

The herein provide composition can further comprise any known or newlydiscovered substance that can be administered to a wound, tissue injury,site of inflammation or cancer. For example, the provided compositioncan further comprise one or more of classes of antibiotics (e.g.Aminoglycosides, Cephalosporins, Chloramphenicol, Clindamycin,Erythromycins, Fluoroquinolones, Macrolides, Azolides, Metronidazole,Penicillin's, Tetracycline's, Trimethoprim-sulfamethoxazole,Vancomycin), steroids (e.g. Andranes (e.g. Testosterone), Cholestanes(e.g. Cholesterol), Cholic acids (e.g. Cholic acid), Corticosteroids(e.g. Dexamethasone), Estraenes (e.g. Estradiol), Pregnanes (e.g.Progesterone), narcotic and non-narcotic analgesics (e.g. Morphine,Codeine, Heroin, Hydromorphone, Levorphanol, Meperidine, Methadone,Oxydone, Propoxyphene, Fentanyl, Methadone, Naloxone, Buprenorphine,Butorphanol, Nalbuphine, Pentazocine), chemotherapy (e.g. anti-cancerdrugs such as but not limited to Altretamine, Asparaginase, Bleomycin,Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cladribine,Cyclophosphamide, Cytarabine, Dacarbazine, Diethylstilbesterol, Ethinylestradiol, Etoposide, Floxuridine, Fludarabine, Fluorouracil, Flutamide,Goserelin, Hydroxyurea, Idarubicin, Ifosfamide, Leuprolide, Levamisole,Lomustine, Mechlorethamine, Medroxyprogesterone, Megestrol, Melphalan,Mercaptopurine, Methotrexate, Mitomycin, Mitotane, Mitoxantrone,Paclitaxel, pentastatin, Pipobroman, Plicamycin, Prednisone,Procarbazine, Streptozocin, Tamoxifen, Teniposide, Vinblastine,Vincristine), anti-inflammatory agents (e.g. Alclofenac; AlclometasoneDipropionate; Algestone Acetonide; alpha Amylase; Amcinafal; Amcinafide;Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac;Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen;Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide;Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate;Clobetasone Butyrate; Clopirac; Cloticasone Propionate; ConnethasoneAcetate; Cortodoxone; Decanoate; Deflazacort; Delatestryl;Depo-Testosterone; Desonide; Desoximetasone; Dexamethasone Dipropionate;Diclofenac Potassium; Diclofenac Sodium; Diflorasone Diacetate;Diflumidone Sodium; Diflunisal; Difluprednate; Diftalone; DimethylSulfoxide; Drocinonide; Endrysone; Enlimomab; Enolicam Sodium;Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole; Fenbufen;Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone;Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate; Flunixin;Flunixin Meglumine; Fluocortin Butyl; Fluorometholone Acetate;Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate;Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; HalopredoneAcetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol;Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole;Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen;Lofemizole Hydrochloride; Lomoxicam; Loteprednol Etabonate;Meclofenamate Sodium; Meclofenamic Acid; Meclorisone Dibutyrate;Mefenamic Acid; Mesalamine; Meseclazone; Mesterolone;Methandrostenolone; Methenolone; Methenolone Acetate; MethylprednisoloneSuleptanate; Momiflumate; Nabumetone; Nandrolone; Naproxen; NaproxenSodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein; Orpanoxin;Oxandrolane; Oxaprozin; Oxyphenbutazone; Oxymetholone; ParanylineHydrochloride; Pentosan Polysulfate Sodium; Phenbutazone SodiumGlycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate; PiroxicamOlamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone;Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex;Salnacedin; Salsalate; Sanguinarium Chloride; Seclazone; Sermetacin;Stanozolol; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate;Talosalate; Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam;Tesimide; Testosterone; Testosterone Blends; Tetrydamine; Tiopinac;Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide;Triflumidate; Zidometacin; Zomepirac Sodium), or anti-histaminic agents(e.g. Ethanolamines (like diphenhydrmine carbinoxamine), Ethylenediamine(like tripelennamine pyrilamine), Alkylamine (like chlorpheniramine,dexchlorpheniramine, brompheniramine, triprolidine), otheranti-histamines like astemizole, loratadine, fexofenadine,Bropheniramine, Clemastine, Acetaminophen, Pseudoephedrine,Triprolidine).

The compositions may be administered topically, orally, or parenterally.For example, the compositions can be administered extracorporeally,intracranially, intravaginally, intraanally, subcutaneously,intradermally, intracardiac, intragastric, intravenously,intramuscularly, by intraperitoneal injection, transdermally,intranasally, or by inhalant. As used herein, “intracranialadministration” means the direct delivery of substances to the brainincluding, for example, intrathecal, intracisternal, intraventricular ortrans-sphenoidal delivery via catheter or needle.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein.

As used herein, “topical intranasal administration” means delivery ofthe compositions into the nose and nasal passages through one or both ofthe nares and can comprise delivery by a spraying mechanism or dropletmechanism, or through aerosolization of the nucleic acid or vector.Administration of the compositions by inhalant can be through the noseor mouth via delivery by a spraying or droplet mechanism. Delivery canalso be directly to any area of the respiratory system (e.g., lungs) viaintubation.

The exact amount of the compositions required will vary from subject tosubject, depending on the species, age, weight and general condition ofthe subject, the severity of the allergic disorder being treated, theparticular nucleic acid or vector used, its mode of administration andthe like. Thus, it is not possible to specify an exact amount for everycomposition. However, an appropriate amount can be determined by one ofordinary skill in the art using only routine experimentation given theteachings herein.

The materials may be in solution or suspension (for example,incorporated into microparticles, liposomes, or cells). These may betargeted to a particular cell type via antibodies, receptors, orreceptor ligands. The following references are examples of the use ofthis technology to target specific proteins to tumor tissue (Senter, etal., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K. D., Br. J.Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703,(1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, etal., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz andMcKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al.,Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as “stealth”and other antibody conjugated liposomes (including lipid mediated drugtargeting to colonic carcinoma), receptor mediated targeting of DNAthrough cell specific ligands, lymphocyte directed tumor targeting, andhighly specific therapeutic retroviral targeting of murine glioma cellsin vivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

Suitable carriers and their formulations are described in Remington: TheScience and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. 1995. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include, but are not limited to,saline, Ringer's solution and dextrose solution. The pH of the solutioncan be from about 5 to about 8, from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of compositionbeing administered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

Pharmaceutical compositions may include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions may also includeone or more active ingredients such as antimicrobial agents,antiinflammatory agents, anesthetics, and the like.

The pharmaceutical composition may be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration may be topically (includingophthalmically, vaginally, rectally, intranasally), orally, byinhalation, or parenterally, for example by intravenous drip,subcutaneous, intraperitoneal or intramuscular injection.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration may include ointments, lotions,creams, gels (e.g., poloxamer gel), drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. The disclosed compositions can be administered, for example,in a microfiber, polymer (e.g., collagen), nanosphere, aerosol, lotion,cream, fabric, plastic, tissue engineered scaffold, matrix material,tablet, implanted container, powder, oil, resin, wound dressing, bead,microbead, slow release bead, capsule, injectables, intravenous drips,pump device, silicone implants, or any bio-engineered materials.

In one aspect the provided pharmaceutically acceptable carrier is apoloxamer. Poloxamers, referred to by the trade name Pluronics®, arenonionic surfactants that form clear thermoreversible gels in water.Poloxamers are polyethylene oxide-polypropylene oxide-polyethylene oxide(PEO-PPO-PEO) tri-block copolymers. The two polyethylene oxide chainsare hydrophilic but the polypropylene chain is hydrophobic. Thesehydrophobic and hydrophilic characteristics take charge when placed inaqueous solutions. The PEO-PPO-PEO chains take the form of small strandswhere the hydrophobic centers would come together to form micelles. Themicelle, sequentially, tend to have gelling characteristics because theycome together in groups to form solids (gels) where water is justslightly present near the hydrophilic ends. When it is chilled, itbecomes liquid, but it hardens when warmed. This characteristic makes ituseful in pharmaceutical compounding because it can be drawn into asyringe for accurate dose measurement when it is cold. When it warms tobody temperature (when applied to skin) it thickens to a perfectconsistency (especially when combined with soy lecithin/isopropylpalmitate) to facilitate proper inunction and adhesion. Pluronic® F127(F127) is widely used because it is obtained easily and thus it is usedin such pharmaceutical applications. F127 has a EO:PO:EO ratio of100:65:100, which by weight has a PEO:PPO ratio of 2:1. Pluronic gel isan aqueous solution and typically contains 20-30% F-127. Thus, theprovided compositions can be administered in F127.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

Effective dosages and schedules for administering the compositions maybe determined empirically, and making such determinations is within theskill in the art. The dosage ranges for the administration of thecompositions are those large enough to produce the desired effect inwhich the symptoms disorder are effected. The dosage should not be solarge as to cause adverse side effects, such as unwantedcross-reactions, anaphylactic reactions, and the like. Generally, thedosage will vary with the age, condition, sex and extent of the diseasein the patient, route of administration, or whether other drugs areincluded in the regimen, and can be determined by one of skill in theart. The dosage can be adjusted by the individual doctor in the event ofany counterindications. Dosage can vary, and can be administered in oneor more dose administrations daily, for one or several days. Guidancecan be found in the literature for appropriate dosages for given classesof pharmaceutical products. The range of dosage largely depends on theapplication of the compositions herein, severity of condition, and itsroute of administration.

For example, in applications as a laboratory tool for research, the ACTpeptide compositions can be used in doses as low as 0.01% w/v. Thedosage can be as low as 0.02% w/v and possibly as high as 2% w/v intopical skin wound treatments. Significantly higher concentrations ofthe compositions by themselves or in combination with other compoundsmay be used in applications like cancer/tumor therapy or as an earlyconcentrated bolus immediately following an acute tissue injury. Thus,upper limits of the provided polypeptides may be up to 2-5% w/v or v/vif given as an initial bolus delivered for example directly into a tumormass. Recommended upper limits of dosage for parenteral routes ofadministration for example intramuscular, intracerebral,intracardicardiac and intraspinal could be up to 1% w/v or v/v dependingon the severity of the injury. This upper dosage limit may vary byformulation, depending for example on how the polypeptide(s) is combinedwith other agents promoting its action or acting in concert with thepolypeptide(s).

For continuous delivery of the provided polypeptides, for example, incombination with an intravenous drip, upper limits of 0.01 g/Kg bodyweight over time courses determined by the doctor based on improvementin the condition can be used. In another example, upper limits ofconcentration of the provided nucleic acids delivered topically, forexample, in skin wounds would be 5-10 μg/cm² of wound depending forexample on how the nucleic acid is combined with other agents promotingits action or acting in concert with the nucleic acids. This would berepeated at a frequency determined by the Doctor based on improvement.In another example, upper limits of concentration of the providednucleic acids delivered internally for example, intramuscular,intracerebral, intracardicardiac and intraspinal would be 50-100 μg/mlof solution. Again, the frequency would be determined by the Doctorbased on improvement.

Also disclosed is the pre-conditioning of an area with the providedpolypeptides prior to surgery. The concentration of the polypeptides canbe 10-200 μM mixed in with 10-30% pluronic gel or any such carrier thatenables penetration of the peptide(s) within the site of interest for aperiod of at least 3-6 hours prior to surgery. This pre-proceduralconditioning can improve the subsequent healing response to surgery,including reduced inflammatory response.

Viral vectors remain highly experimental tools that nonetheless showconsiderable potential in clinical applications. As such, caution iswarranted in calculation of expected dosage regimes for viral vectorsand will depend considerably on the type of vector used. For example,retroviral vectors infect dividing cells such as cancer cellsefficiently, intercalating into the host cell genome and continuingexpression of encoded proteins indefinitely. Typical dosages ofretroviruses in an animal model setting are in the range of 10⁷ to 10⁹infectious units per ml. By contrast, adenoviruses most efficientlytarget post-mitotic cells, but cells are quickly eliminated by the hostimmune system or virus is eventually lost if infected cells resumeproliferation and subsequently dilute the viral episomal DNA. Indeed,this transient time course of infection may be useful for short-termdelivery of the composition described herein in certain clinicalsituations, for example in amelioration of a small injury. In animalmodels, concentrations of 10⁸-10¹¹ infectious units per ml of adenovirusare typical for uses in research. Dose ranges of vectors based on dataderived from animal models would be envisaged to be used eventually inclinical setting(s), pending the development of pharmaceuticallyacceptable formulation(s).

Two topical applications of ACT compositions at 0.02% w/v; one appliedacutely and the second applied 24 hours later are sufficient to reduceinflammation, promote healing, reduce scarring, increase tensilestrength, and promote tissue regeneration. However, in a clinicalsetting an increased frequency of up to 3 applications per day topicallyat a concentration of up to 5% is recommended until significantimprovement is achieved as determined by a Doctor. For internaladministration, for example, intravenously, intramuscularly,intracerebral, intracardicardiac and intraspinally and increasedfrequency of up to 3 dosages of 1% w/v or v/v per day is recommendeduntil significant improvement is determined by the Doctor.

Following administration of a disclosed composition, such as apolypeptide, for promoting wound healing, the efficacy of thetherapeutic composition can be assessed in various ways well known tothe skilled practitioner. For instance, one of ordinary skill in the artwill understand that a composition, such as a polypeptide, disclosedherein is efficacious in promoting wound healing in a subject byobserving that the composition can reduce scar tissue formation, reducefibrotic tissue formation, improve tissue regeneration, or reduceinflammation in the subject following tissue injury. Methods formeasuring these criteria are known in the art and discussed herein.

Also provided are materials comprising the herein provided compositions(e.g., polypeptides, nucleic acids, or vectors). For example, providedare materials used to treat wounds, wherein the materials are coatedwith an ACT polypeptide. Non-limiting examples of materials used totreat wounds include bandages, steri-strip, sutures, staples, or grafts(e.g., skin grafts).

For example, the material (e.g., bandage, steri-strip, suture, staple,graft) can be soaked in the provided polypeptide at a concentrationranging from 10-200 μM. The material can then be dried and sealed in asterile container. The material can also be immersed in liquid 10-30%pluronic gel at 4° C. containing polypeptide at 10-200 μM concentration.The material can then be brought to approximate room temperature so thatthe gel polymerizes, leaving a coat of polypeptide-impregnated gelsurrounding the material, which can be sealed in a sterile container.The polypeptide can also be incorporated into a cross-linkable hydrogelsystem, such as the poly(lactic-co-glycolic acid) (PLGA) orpolyurethane, which can then be fashioned into materials for treatingwounds (e.g., bandage, steri-strip, suture, staple, graft). Thus,provided are composite hydrogel-peptide materials.

Also disclosed are medical implants coated with the provided polypeptidebefore implantation in a subject. For example, a common problem in suchimplant surgeries is the formation of a contraction capsule around theimplant from scar tissue formation that leads to undue hardening,contraction and ultimately misshaping of the tissue of interest. The useof the present polypeptides in or on the implant can reduce or preventthis misshaping. Non-limiting examples of medical implants include: limbprostheses, breast implants, penile implants, testicular implants,artificial eyes, facial implants, artificial joints, heart valveprostheses, vascular prostheses, dental prostheses, facial prosthesis,tilted disc valve, caged ball valve, ear prosthesis, nose prosthesis,pacemakers, cochlear implants, and skin substitutes (e.g., porcineheterograft/pigskin, BIOBRANE, cultured keratinocytes).

A. METHODS

Provided herein is a method of promoting wound healing following tissueinjury in a subject, comprising administering to the subject one or moreof the herein provided compositions (e.g., polypeptides, nucleic acids,or vectors) in a pharmaceutically acceptable carrier. Further providedis a method of treating a subject with tissue injury, comprisingadministering to the subject one or more of the herein providedcompositions (e.g., polypeptides, nucleic acids, or vectors) in apharmaceutically acceptable carrier.

“Promote,” “promotion,” and “promoting” refer to an increase in anactivity, response, condition, disease, or other biological parameter.This can include but is not limited to the initiation of the activity,response, condition, or disease. This may also include, for example, a10% increase in the activity, response, condition, or disease ascompared to the native or control level. Thus, the increase can be a 10,20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of increase inbetween as compared to native or control levels.

By “treat” or “treatment” is meant a method of reducing the effects of adisease or condition. Treatment can also refer to a method of reducingthe underlying cause of the disease or condition itself rather than justthe symptoms. The treatment can be any reduction from native levels andcan be but is not limited to the complete ablation of the disease,condition, or the symptoms of the disease or condition. For example, adisclosed method for promoting wound healing is considered to be atreatment if there is a 10% reduction in one or more symptoms of thedisease in a subject with the disease when compared to native levels inthe same subject or control subjects. Thus, the reduction can be a 10,20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction inbetween as compared to native or control levels.

As used herein, “subject” includes, but is not limited to, animals,plants, bacteria, viruses, parasites and any other organism or entitythat has nucleic acid. The subject may be a vertebrate, morespecifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep,goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a birdor a reptile or an amphibian. The subject can be an invertebrate, morespecifically an arthropod (e.g., insects and crustaceans). The term doesnot denote a particular age or sex. Thus, adult and newborn subjects, aswell as fetuses, whether male or female, are intended to be covered. Apatient refers to a subject afflicted with a disease or disorder. Theterm “patient” includes human and veterinary subjects.

The provided method can reduce scar tissue formation in a subjectfollowing tissue injury. By “scar tissue” is meant the fibrous(fibrotic) connective tissue that forms at the site of injury or diseasein any tissue of the body, caused by the overproduction of disorganizedcollagen and other connective tissue proteins, which acts to patch thebreak in the tissue. Scar tissue may replace injured skin and underlyingmuscle, damaged heart muscle, or diseased areas of internal organs suchas the liver. Dense and thick, it is usually paler than the surroundingtissue because it is poorly supplied with blood, and although itstructurally replaces destroyed tissue, it cannot perform the functionsof the missing tissue. It is composed of collagenous fibers, which willoften restrict normal elasticity in the tissue involved. Scar tissue maytherefore limit the range of muscle movement or prevent propercirculation of fluids when affecting the lymphatic or circulatorysystem. Glial scar tissue following injury to the brain or spinal chordis one of the main obstacles to restoration of neural function followingdamage to the central nervous system. A reduction in scar tissue can beassessed by the population of cell types within the injured site. Forexample, a reduction in glial scar tissue can be estimated by anincreased ratio of neuronal to astrocytic cells. A reduction in scartissue formation can be measured by a simple measurement of scar widthor area of scar tissue (Wilgus et al., 2003). In addition histologicalassessments can be made about the restoration of structural complexitywithin healed tissue in comparison to normal tissue.

In addition to reducing fibrotic tissue formation in a subject infollowing tissue injury, the provided compositions and methods can alsobe used to treat disorders associated with pathological increases infibrotic tissue formation in a subject, such as for example, psoriasis,cutaneous and systemic mastocytosis, asthma, eczema, sinusitis,atherosclerosis, rheumatoid arthritis, inflammatory bowel disease,multiple sclerosis, pulmonary fibrosis and cystic fibrosis. A reductionin fibrotic tissue formation in a subject can be measured by clinicaljudgment of a doctor assessing whether a regain in normal structure andfunction of a given tissue and/or organ in a subject has resultedfollowing a treatment. As an example, for psoriasis a doctor wouldassess the subject's skin to determine whether there has been areduction in patches of raised red skin covered by flaky white buildup.Certain kinds of psoriasis, are characterized by a pimple-ish (pustularpsoriasis) or burned (erythrodermic) appearance. In such cases, thedoctor would determine whether treatment has resulted in the reductionof these symptoms. In the case of an tissue or organ in which a subjectwhere a doctor judges that a biopsy is clinically available and/ornecessary or in an animal model of the human disease, tissue fragmentsof biopsies would be prepared and tissue histological structure would beassessed by a clinical pathologist and/or trained histopathologist todetermine if reduction in fibrosis and restoration of normal tissuestructure and function has occurred. The area of fibrosis to normaltissue could also be quantitatively assessed on such histologicalpreparations.

The provided method can restore normal tissue mechanical properties suchas tensile strength following tissue injury in a subject. “Tensilestrength” refers to the amount of stress or strain required to break thetissue or wound.

The tensile strength of treated wounds can be 60, 65, 70, 75, 80, 85,90, 95, 100% that of uninjured tissue within 3 months after treatment.Thus, provided is a method of restoring tissue mechanical properties,including increasing tensile strength of a healed injury to approach orreach that of normal uninjured tissue, in a subject comprisingadministering to the subject one or more of the herein providedcompositions (e.g., polypeptides, nucleic acids, or vectors) in apharmaceutically acceptable carrier.

The type of wounds that would be important with respect to tensilestrength/extensibility would include injuries to musculoskeletalstructures/tissues, and the skin covering these structures. For example,the provided methods can improve tensile strength of articulatingjoints, bone, cartilage, tendons, or ligaments. The provided methods canalso improve tensile strength of skin under higher degrees ofstress/strain, such as the skin covering the elbow, knee, or foot. Themost common problems associated with healing of joint injuries is thatexcessive scarring in these areas leads to contraction, andnon-extensibility of the healed joint area. This has serious cosmeticand psychological consequences. The properties of the peptides will helpmodulate and lessen the formation of such scar tissue leading to greatermobility of the joint.

The provided method can improve tissue regeneration following tissueinjury in a subject. By “regeneration” is meant the renewal, re-growth,or restoration of a body or a bodily part, tissue, or substance afterinjury or as a normal bodily process. In contrast to scarring, tissueregeneration involves the restoration of the tissue to its originalstructural, functional, and physiological condition. This is alsoreferred to herein as tissue “complexity”. The restoration can bepartial or complete, meaning 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%restoration, or any amount of restoration in between as compared tonative or control levels. As an example, in the case of a skin injury,tissue regeneration can involve the restoration of hair follicles,glandular structures, blood vessels, muscle, or fat. In the case of abrain injury, tissue regeneration can involve maintenance or restorationof neurons. As an example in the case of skin an improvement in tissueregeneration can be assessed by measurements of the volume of fibrousscar tissue to normal regenerated skin as a ratio. As another example,counts can be made of discrete regenerating structures such asregenerating skin glands normalized to the volume of the wound area.

In one aspect, tissue regeneration involves the recruitment anddifferentiation of stem cells to replace the damaged cells. As usedherein, a “stem cell” is an undifferentiated cell found amongdifferentiated cells in a tissue or organ, or introduced from anexternal source for e.g., Embryonic stem cells, Adult Bone Marrow stemcells, that can renew itself and differentiate to yield the majorspecialized cell types of the tissue or organ. The primary roles of stemcells in a living organism are to maintain and repair the tissue inwhich they are found. By stem cell differentiation is meant the processwhereby an unspecialized cell (e.g., stem cell) acquires the features ofa specialized cell such as a skin, neural, heart, liver, or muscle cell.As an example, in the case of a skin injury, tissue regeneration caninvolve the differentiation of stem cells present in the epithelium intohair follicles (Alonso and Fuchs, 2003). In the case of a brain injury,tissue regeneration can involve the differentiation of stem cells intoneurons. The provided method can enhance stem cell differentiationfollowing tissue injury in a subject. Enhanced stem cell differentiationcan be measured by providing a clinically acceptable genetic or othermeans of marking endogenous or engrafted stem cells and determining thefrequency of differentiation and incorporation of marked stem cells intonormal tissue structures. As another example, certain structures such ashair follicles are known to be regenerated from endogenous stem cellsfollowing tissue injury. As such, counts of hair follicles normalized totissue injury area would serve as a quantitative assessment of enhancedstem cell differentiation.

The provided method can reduce inflammation in a subject. By“inflammation”, “inflammatory response” or “immune response” is meantthe reaction of living tissues to injury, infection or irritationcharacterized by redness, warmth, swelling, pain, and loss of function,produced as the result of increased blood flow and an influx of immunecells and secretions. Inflammation is the body's reaction to invadinginfectious microorganisms and results in an increase in blood flow tothe affected area, the release of chemicals that draw white blood cells,an increased flow of plasma, and the arrival of monocytes (or astrocytesin the case of the brain) to clean up the debris. Anything thatstimulates the inflammatory response is said to be inflammatory. Thus,in addition to reducing inflammation in a subject in response to tissueinjury, the provided compositions and methods can also be used to treatdisorders associated with pathological increases in levels ofinflammatory cells, including, for example, asthma, eczema, sinusitis,atherosclerosis, rheumatoid arthritis, inflammatory bowel disease,cutaneous and systemic mastocytosis, psoriasis, and multiple sclerosis.Treatment with the provided polypeptide can also reduce itching, forexample of healing wounds. Generally, itching results from histaminerelease by mast cells. The provided polypeptide can reduce mast cellde-granulation and histamine release. Thus, the provided polypeptide canbe used to treat conditions involving histamine release, including, butnot limited to, itching, scratching, sinus irritation, allergic cough,red eyes, asthma, and eczema.

A reduction in inflammation can be measured by a reduction in thedensity of inflammatory cell types such as, for example, monocytes orastrocytes. A reduction in inflammation can be measured by a reductionin the density of inflammatory cell types such as, for example,neutrophils, mast cells, basophils, and monocytes. A reduction ininflammation can be calculated by an in vivo measurement of neutrophilactivity (Jones et al., 1994). In addition factors like frequency ofmast cell degranulation or measurement of histamine levels or levels ofreactive oxygen species can be used as measurements of reduction ininflammation. The level of inflammation can also be indirectly measuredby checking for transcription levels of certain genes by qRT-PCR fore.g. genes like, Interferon-alpha, -beta and -gamma, Tumor NecrosisFactor-alpha, Interleukine 1beta, -2, -4, -5, -6, -8, -12, -18, -23,-27, CD4, CD28, CD80, CD86, MHCII, and iNOS. Measurement ofpro-inflammatory cytokine levels in the tissues and or bodily fluids ofthe subject including plasma can measure a reduction in inflammation. Itis noteworthy that a mechanism of ACT peptide action may be byinhibition of inflammatory cell migration and/or inhibition ofpro-inflammatory chemicals (histamine, reactive oxygen species) andpro-inflammatory cytokines such as Interleukin (IL)-1, IL-6, IL-8 andtumor necrosis factor (TNF).

The provided method can inhibit proliferation of a transformed cell in asubject (see FIG. 2). By transformed cell is meant a neoplasm, cancer,or tumor cell that divides and reproduces abnormally with uncontrolledgrowth. Thus, inhibition of proliferation (i.e., hyperplasia) of saidtransformed cell results in a reduction in the growth and thusmalignancy of the cancer. A representative but non-limiting list ofcancers that the disclosed compositions and methods can be used to treatis the following: glioma, lymphoma, B cell lymphoma, T cell lymphoma,mycosis fungoides, Hodgkin's Disease, myeloid leukemia, bladder cancer,brain cancer, nervous system cancer, head and neck cancer, squamous cellcarcinoma of head and neck, kidney cancer, lung cancers such as smallcell lung cancer and non-small cell lung cancer, neuroblastoma,glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skincancer, liver cancer, melanoma, squamous cell carcinomas of the mouth,throat, larynx, and lung, colon cancer, cervical cancer, cervicalcarcinoma, breast cancer, and epithelial cancer, renal cancer,genitourinary cancer, pulmonary cancer, esophageal carcinoma, head andneck carcinoma, large bowel cancer, hematopoietic cancers, testicularcancer, colon and rectal cancers, prostatic cancer, or pancreaticcancer. Thus, the provided method can be used to treat cancer in asubject. For example, the provided method can be used to treat glioma ina subject.

An inhibition in transformed cell proliferation can be measured by avariety of cell proliferation markers and kits for e.g. Ki67/MIB-1immunostaining, tritiated thymidine or bromodeoxyuridine labelingindices, DNA S-phase fraction, proliferating cell nuclear antigenexpression, potential doubling time and analysis of the nucleolarorganizer region associated proteins (AgNORs). Since the proliferativeactivity of the tumor depends both on the proportion of cells committedto the cycle (growth fraction) and the speed of the cell cycle, theactual proliferative activity of a tumor could well be measured by theequation [PA=Ki67 or MIB-1 scores×AgNORs] (Pich et al., 2004). Inanother example, histopathologists are skilled in assessing biopsytissue sections using simple qualitative and quantitative indices ofmitosis to determine proliferation in transformed cell populations

Various mouse models have been developed for cancer research. There arespecific mouse models for specific types of cancers. For example,Bladder cancer, Cervical cancer, Endometrial cancer, Gastrointestinalcancer, Genitourinary cancer, Head and Neck cancer, Hematopoieticcancer, Kidney cancer, Lung cancer, Mammary Gland cancer, Melanoma,Myeloma, Nervous System cancer, Oral cancer, Ovarian cancer, Pancreaticcancer, Prostate cancer, Sarcoma, Skin cancer. These models are welldescribed and used. The favorable effects of the polypeptides, nucleicacids or vectors provided herein can be studied in any of these models.For example the skin cancer mouse model can be easily used fordemonstration. Cancers can be cultivated applying the xenograft model ofgrowing human cancerous tissues using the specific pathogen free, homoinbred mouse (a nude mouse) (Yoo, 2004). The polypeptides, nucleic acidsor vectors provided herein can be locally administered for e.g.bioengineered materials such as a hollow fiber membranes (Orlandini andMargaria. 1983; Ming Chu et al., 1998) and microfibers, slow releasebeads, hypodermic needles, indwelling catheters, which can be insertedlocally into the cancerous growth, or systemically administered to reachits target for e.g. intravenous infusions, intramuscularly,intraperitoneal injection. This treatment can be administered by itselfor in combination with other therapeutic compounds for e.g.Chemotherapeutic agents.

The provided method can inhibit metastasis of a transformed cell in asubject. By “metastasis” is meant the transmission of cancer cells froman original site to one or more sites elsewhere in the body, usually byway of the blood vessels or lymphatics. Metastatis can be broken downinto a series of events. First, cancer cell migration begins the processby which tumor cells leave the primary site of growth, often penetratingthe basement membrane and moving towards the local vasculature.Intravasation describes the process of cancer cell entry into thevasculature, and distribution to distant sites. Extravasation refers tothe process of cancer cell egression from the vasculature. Finally,proliferation of cancer cells at the distant site is profoundlyinfluenced by localized growth factor availability, influences ofstromal cells, and the surrounding extracellular matrix milieu (theso-called “soil”) as well as the availability of nutrients and factorsprovided by the resultant vascularization of the growing tumor. Thus,the provided compositions and methods can inhibit metastasis of atransformed cell in a subject by inhibiting migration (i.e., metastaticmigration) of said cell. Tumourigenesis is the result of cell cycledisorganization, leading to an uncontrolled cellular proliferation.Specific cellular processes-mechanisms that control cell cycleprogression and checkpoint traversation through the intermitotic phasesare deregulated. Normally, these events are highly conserved due to theexistence of conservatory mechanisms and molecules such as cell cyclegenes and their products. An inhibition in metastatic migration can bemeasured by the levels of such cell cycle genes and products for e.g.cyclins, cyclin dependent kinases (Cdks), Cdk inhibitors (CKI) and extracellular factors (i.e. growth factors). Revolutionary techniques usinglaser cytometry and commercial software are available to quantify andevaluate cell cycle processes and cellular growth. S-phase fractionmeasurements, including ploidy values, using histograms and estimationof indices such as the mitotic index and tumour-doubling time indices,provide adequate information to the clinician to evaluate tumouraggressiveness.

As used herein, tissue injury can result from, for example, a scrape,cut, laceration wound, crush wound, compression wound, stretch injury,bite wound, graze, bullet wound, explosion injury, body piercing, stabwound, burn wound, wind burn, sun burn, chemical burn, surgical wound,surgical intervention, medical intervention, host rejection followingcell, tissue or organ grafting, pharmaceutical effect, pharmaceuticalside-effect, bed sore, radiation injury, cosmetic skin wound, internalorgan injury, disease process (e.g., asthma, cancer), infection,infectious agent, developmental process, maturational process (e.g.,acne), genetic abnormality, developmental abnormality, environmentaltoxin, allergen, scalp injury, facial injury, jaw injury, foot injury,toe injury, finger injury, bone injury, sex organ injury, joint injury,excretory organ injury, eye injury, corneal injury, muscle injury,adipose tissue injury, lung injury, airway injury, hernia, anus injury,piles, ear injury, retinal injury, skin injury, abdominal injury, arminjury, leg injury, athletic injury, back injury, birth injury,premature birth injury, toxic bite, sting, tendon injury, ligamentinjury, heart injury, heart valve injury, vascular system injury,cartilage injury, lymphatic system injury, craniocerebral trauma,dislocation, esophageal perforation, fistula, nail injury, foreign body,fracture, frostbite, hand injury, heat stress disorder, laceration, neckinjury, self mutilation, shock, traumatic soft tissue injury, spinalcord injury, spinal injury, sprain, strain, tendon injury, ligamentinjury, cartilage injury, thoracic injury, tooth injury, trauma, nervoussystem injury, aging, aneurism, stroke, digestive tract injury, infarct,or ischemic injury.

The peptides and/or other formulations embodying the invention willmodulate cell migration and proliferation, thereby reducinginflammation, accelerating wound healing, reduce scarring and ultimatelypromote repair, regeneration and restoration of structure and functionin all tissues. Healing of wounds, post-peptide application will involvesignificantly reduced fibrosis, consequently reduced scarring in skinwounds and fibrous patches in internal tissue injuries, promoting tissueregeneration and restoring tissue and organ structure and function. Anadditional embodiment of the invention comprises an in vitro scratchwound assay of cell migration that the peptide alters and modulatesmigration and proliferation of various cultured cell types, includingbut not limited to, fibroblasts/mesenchymal cells, tumor cells andepithelial cells.

Further, said peptides and/or formulations embodying the invention canbe used to treat external wounds caused by, but not limited to scrapes,cuts, lacerated wounds, bite wounds, bullet wounds, stab wounds, burnwounds, sun burns, chemical burns, surgical wounds, bed sores, radiationinjuries, all kinds of acute and chronic wounds, wounds or lesionscreated by cosmetic skin procedures and also ameliorate the effects ofskin aging. The actions of said peptides and/or other formulations willaccelerate wound healing in all kinds of external wounds and improve thecosmetic appearance of wounded areas, and skin subject to aging anddisease. Said peptides and/or other formulations can be used to treatinternal injury caused by, but not limited to, disease, surgery,gunshots, stabbing, accidents, infarcts, ischemic injuries, to organsand tissues including but not limited to heart, bone, brain, spinalcord, retina, peripheral nerves and other tissues and organs commonlysubject to acute and chronic injury, disease, congenital anddevelopmental malformation and aging processes. Injury to internalorgans causes a fibrotic response, which leads to loss of structure andfunction in organ systems. In central nervous system (CNS) this responseto injury is mediated by astrocytes (fibroblast-like cells in the CNS)and thus will subsequently be referred to as an astrocytic response.Embodiments of our invention will alleviate this fibrotic/astrocyticresponse hence helping in repair and regeneration of injured tissues andrestoration of tissue and organ structure and function.

Further embodiments of the inventions include the use of said peptidesand/or other formulations to improve angiogenesis by stimulatingangiogenic factors like, but not limited to VEGF, and improvedifferentiation of vascular tissues thereby improving blood flow to thesite of tissue injury.

Increased blood supply to the wound site stimulated by our treatmentswill result in reduced scarring in external and internal wounds andpromote improved repair and regeneration of tissues and organs.

Additional embodiments of the invention comprises the use of saidpeptides and/or other formulations for tissue and organ regeneration,when administered in association with stem cells and/or drugs and/orother endogenous and/or clinical regimens promoting stem cellmobilization and/or tissue regeneration. Stern cells will help in tissueregeneration and our treatment will promote differentiation directlyand/or indirectly by processes that include, but are not limited toreduced fibrotic/astrocytic scar formation, thereby restoring normaltissue structure and function. Our treatment will promote the generationof a permissive environment in vivo for regeneration and restoration ofstructure and function of tissues and organs. Regenerative processesaided by our peptide include, but are not limited to internal andexternal injury, regeneration of tissues, organs, or other body parts,healing and restoration of function following vascular occlusion andischemia, brain stroke, myocardial infarction, spinal cord damage, braindamage, peripheral nerve damage, retinal damage, bone damage and otherinsults to tissues causing destruction, damage or otherwise resultingfrom, but not limited to, injury, surgery, cancer, congenital anddevelopmental malformation, and diseases causing progressive loss oftissue structure and function, including but not limited to diabetes,bacterial, viral and prion-associated diseases, Alzheimer's disease,Parkinson's disease, AIDs and other genetically determined,environmentally determined or idiopathic disease processes causing lossof tissue/organ/body part structure and function. In addition, we claimthat our peptide can be administered with drugs or other compoundspromoting tissue and cellular regeneration including, but not limitedto, trophic factors in processes including, but not limited to, brain,retina, spinal cord and peripheral nervous system regeneration (e.g.,NGFs, FGFs, Neurotrophins, Neuregulins, Endothelins, GDNFs, BDNF, BMPs,TGFs, Wnts).

Said peptides and/or other formulations can be used in bioengineeringapproaches to tissue and organ repair, regeneration and restoration ofstructure and function, including but not limited to, application withbioengineered delivery vehicles. These include but are not limited to,nanoparticles, fibers, gels, polymers, polyethylene glycol and otherbioengineered materials designed for the purpose of promoting tissuerepair and/or targeted and/or sustained release of our peptide andtissue scaffolds, polymer matrices and other bioengineered surfaces orstructures coated or otherwise treated to release, maintain or localizethe effects of said peptides and/or other formulations in associationwith the other beneficial effects or otherwise of these bioengineeredmaterials.

Additional embodiments of the invention comprise the use of saidpeptides and/or other formulations in vitro and/or in animal modelshumanized or otherwise to promote and/or assist in the regeneration oftissues, organs and body parts for use, but not limited to organ/tissueor body part transplantation.

A further embodiment of the invention comprises the use of said peptidesand/or other formulations to alleviate the symptoms of MultipleSclerosis (MS). MS is a chronic disease of the central nervous system.Pathologically, MS is characterized by the presence of areas ofdemyelination and T-cell predominant perivascular inflammation in thebrain white matter. The anti-inflammatory and regenerative properties ofour treatment will help in the treatment of MS and other conditionssimilar to it. Said peptide will help with conditions like, but notlimited to psoriasis, scleroderma, acne, eczema and other diseases ofskin and connective tissues. Psoriasis, is a chronic, inflammatory skindisease characterized by an uncontrolled shedding of the skin andafflicts millions of people throughout the world. The effects of ourtreatment on fibroblasts and inflammatory response of the treatments, asstated in the claims, will help alleviate Psoriasis. Eczema ischaracterized by painful swelling, oozing of the skin, bleeding cracks,severe scaling, itching and burning. As stated above, the effects of ourtreatment on fibroblasts and inflammatory response, combined withaccelerated healing properties will relieve symptoms of eczema.

Said peptides and/or other formulations will help with repair aftercosmetic and/or clinical procedures involving, but not limited to,controlled damage—e.g., corneal laser surgery, laser anddermabrasion/dermaplaning, skin resurfacing, and punch excision.Application of our treatment immediately after surgery or any cosmeticprocedure will reduce or eliminate scarring. Uses of said peptidesand/or other formulations will reduce keloid scar formation. Keloidscars are common in dark skin people of Asian, African, or MiddleEastern descent. Keloid scar is a thick, puckered, itchy cluster of scartissue that grows beyond the edges of a wound or incision. Keloid scarsare sometimes very nodular in nature, and they are often darker in colorthan surrounding skin. They occur when the body continues to producetough, fibrous protein (known as collagen) after a wound has healed.Application of our treatment will ameliorate formation of these Keloidscars.

Additional uses of said peptides and/or other formulations will helpcorrect other diseases and other conditions (e.g., congenital anddevelopmental defects, aging) associated with inflammatory response,fibrosis and connective tissue disorders. Fibrosis is a common conditionnoted after trauma to any bodily organ or tissue. Excessive fibrosisresults in loss of structure and function and scarring at the traumasite. Our treatment will reduce fibrosis and promote regeneration, andrestoration of structure and function.

Said peptides and/or other formulations will modulate cell proliferationand can be used alone or in association with drugs used in the treatmentof uncontrolled proliferation (e.g., anticancer drugs) and procedures(e.g., radiation therapy). Diseases of uncontrolled cell proliferation,or hyperplasias, are common health problems. Examples of diseases ofcell over-proliferation include but are not limited to psoriasis,seborrhea, scleroderma, eczema, benign prostate hyperplasia, congenitaladrenal hyperplasia, endometrial hyperplasia, squamous cell (vulvular)hyperplasia, sebaceous hyperplasia, Crohn's Disease, leukemia,carcinoma, sarcoma, glioma, and lymphoma. Our peptides limit undesirablecellular proliferation and will thus improve prognosis of conditionsassociated with excessive cell proliferation.

Said peptides and/or other formulations will have effects on cellmigration, proliferation and differentiation and thus will assist inpreventing metastasis. Said peptides and/or other formulations can beadministered alone or in association with drugs or procedures used inthe treatment of metastasis like but not limited to, Altretamine,Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine,Chlorambucil, Cisplatin, Cladribine, Cyclophosphamide, Cytarabine,Dacarbazine, Diethylstilbesterol, Ethinyl estradiol, Etoposide,Floxuridine, Fludarabine, Fluorouracil, Flutamide, Goserelin,Hydroxyurea, Idarubicin, Ifosfamide, Leuprolide, Levamisole, Lomustine,Mechlorethamine, Medroxyprogesterone, Megestrol, Melphalan,Mercaptopurine, Methotrexate, Mitomycin, Mitotane, Mitoxantrone,Paclitaxel, pentastatin, Pipobroman, Plicamycin, Prednisone,Procarbazine, Streptozocin, Tamoxifen, Teniposide, Vinblastine,Vincristine. Metastasis is the spread of cancer from its primary site toother places in the body. Cell migration is the movement of cells fromone part of the body to another. Our treatments effects on cellmigration demonstrates its ability to inhibit spread of tumors.

Additional embodiments of the invention comprise the delivery of saidpeptides and/or other formulations using techniques such as, but notlimited to, all Antennapedia sequences, and related cell internalizationvectors (e.g., TAT protein transduction domain, all TAT peptides, allTAT fusion proteins), viral gene delivery vectors, DNA expressionvectors, and any other delivery method that can help get our peptide tothe tissue and/or cellular site of action by itself or in associationwith other agents including but not limited to co-factors assisting thisdelivery (e.g., including but limited to TAT-HA2) and/or stem cells,drugs and other formulations which help in repair, regeneration andrestoration of organ and tissue structure and function.

B. METHODS OF MAKING THE COMPOSITIONS

The compositions disclosed herein and the compositions necessary toperform the disclosed methods can be made using any method known tothose of skill in the art for that particular reagent or compound unlessotherwise specifically noted.

For example, the provided nucleic acids can be made using standardchemical synthesis methods or can be produced using enzymatic methods orany other known method. Such methods can range from standard enzymaticdigestion followed by nucleotide fragment isolation (see for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989)Chapters 5, 6) to purely synthetic methods, for example, by thecyanoethyl phosphoramidite method using a Milligen or Beckman System1Plus DNA synthesizer (for example, Model 8700 automated synthesizer ofMilligen-Biosearch, Burlington, Mass. or ABI Model 380B). Syntheticmethods useful for making oligonucleotides are also described by Ikutaet al., Ann. Rev. Biochem. 53:323-356 (1984), (phosphotriester andphosphite-triester methods), and Narang et al., Methods Enzymol.,65:610-620 (1980), (phosphotriester method). Protein nucleic acidmolecules can be made using known methods such as those described byNielsen et al., Bioconjug. Chem. 5:3-7 (1994).

One method of producing the disclosed polypeptides, such as SEQ ID NO:2,is to link two or more peptides or polypeptides together by proteinchemistry techniques. For example, peptides or polypeptides can bechemically synthesized using currently available laboratory equipmentusing either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc(tert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., FosterCity, Calif.). One skilled in the art can readily appreciate that apeptide or polypeptide corresponding to the disclosed proteins, forexample, can be synthesized by standard chemical reactions. For example,a peptide or polypeptide can be synthesized and not cleaved from itssynthesis resin whereas the other fragment of a peptide or protein canbe synthesized and subsequently cleaved from the resin, thereby exposinga terminal group which is functionally blocked on the other fragment. Bypeptide condensation reactions, these two fragments can be covalentlyjoined via a peptide bond at their carboxyl and amino termini,respectively, to form a protein, or fragment thereof. (Grant G A (1992)Synthetic Peptides: A User Guide. W.H. Freeman and Co., N.Y. (1992);Bodansky M and Trost B., Ed. (1993) Principles of Peptide Synthesis.Springer-Verlag Inc., NY (which is herein incorporated by reference atleast for material related to peptide synthesis). Alternatively, thepeptide or polypeptide is independently synthesized in vivo as describedherein. Once isolated, these independent peptides or polypeptides may belinked to form a peptide or fragment thereof via similar peptidecondensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segmentsallow relatively short peptide fragments to be joined to produce largerpeptide fragments, polypeptides or whole protein domains (Abrahmsen L etal., Biochemistry, 30:4151 (1991)). Alternatively, native chemicalligation of synthetic peptides can be utilized to syntheticallyconstruct large peptides or polypeptides from shorter peptide fragments.This method consists of a two step chemical reaction (Dawson et al.Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779(1994)). The first step is the chemoselective reaction of an unprotectedsynthetic peptide-thioester with another unprotected peptide segmentcontaining an amino-terminal Cys residue to give a thioester-linkedintermediate as the initial covalent product. Without a change in thereaction conditions, this intermediate undergoes spontaneous, rapidintramolecular reaction to form a native peptide bond at the ligationsite (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I etal., J. Biol. Chem., 269:16075 (1994); Clark-Lewis I et al.,Biochemistry, 30:3128 (1991); Rajarathnam K et al., Biochemistry33:6623-30 (1994)).

Alternatively, unprotected peptide segments are chemically linked wherethe bond formed between the peptide segments as a result of the chemicalligation is an unnatural (non-peptide) bond (Schnolzer, M et al.Science, 256:221 (1992)). This technique has been used to synthesizeanalogs of protein domains as well as large amounts of relatively pureproteins with full biological activity (deLisle Milton R C et al.,Techniques in Protein Chemistry IV. Academic Press, New York, pp.257-267 (1992)).

Disclosed are processes for making the compositions as well as theintermediates leading to the compositions. There are a variety ofmethods that can be used for making these compositions, such assynthetic chemical methods and standard molecular biology methods. It isunderstood that the methods of making these and the other disclosedcompositions are specifically disclosed. Disclosed are nucleic acidmolecules produced by the process comprising linking in an operative waya nucleic acid encoding a polypeptide disclosed herein and a sequencecontrolling the expression of the nucleic acid. Disclosed are cellsproduced by the process of transforming the cell with any of the hereindisclosed nucleic acids. Disclosed are any of the disclosed peptidesproduced by the process of expressing any of the herein disclosednucleic acids. Disclosed are animals produced by the process oftransfecting a cell within the animal with any of the nucleic acidmolecules disclosed herein. Disclosed are animals produced by theprocess of transfecting a cell within the animal any of the nucleic acidmolecules disclosed herein, wherein the animal is a mammal. Alsodisclosed are animals produced by the process of transfecting a cellwithin the animal any of the nucleic acid molecules disclosed herein,wherein the mammal is mouse, rat, rabbit, cow, sheep, pig, or primate.Also disclose are animals produced by the process of adding to theanimal any of the cells disclosed herein.

C. KITS

The materials described above as well as other materials can be packagedtogether in any suitable combination as a kit useful for performing, oraiding in the performance of, the disclosed method. It is useful if thekit components in a given kit are designed and adapted for use togetherin the disclosed method. For example disclosed are kits for promotingwound healing, the kit comprising one or more of the polypeptides,nucleic acids or vectors provided herein in a pharmaceuticallyacceptable carrier. Such kits can also include gels, bandages, Milliporetapes, Medicated Q-tips, Sprays, Drops, Syrups, Liquids, Disposabletubes or pouches. The kits also can contain instructions for proper useand safety information of the product or formulation. The kits maycontain dosage information based on the application and method ofadministration as determined by a doctor.

D. USES

The disclosed methods and compositions are applicable to numerous areasincluding, but not limited to, laboratory research tools. Theseformulations play regulatory roles in several cellular processes fore.g. Cell Proliferation, Cell Migration. These formulations can be usedin the laboratory in both in vitro and in vivo model systems forstudying various cellular processes, cell cycle regulations, cellbehavior, responses of cells, organs or tissues to test compounds etc.The formulations can be supplied by themselves or in combination withother compounds or as part of a kit, such as a kit for cellproliferation assay. The kit may contain the formulations mentionedherein by themselves or in combination with other compounds. Such a kitwould include instructions designed to facilitate the experiment. Otheruses are disclosed, apparent from the disclosure, and/or will beunderstood by those in the art.

EXAMPLES Example 1: In Vitro Scratch Injury

Myocytes from neonatal rat hearts were grown until forming anear-confluent monolayer on a tissue culture dish according to standardprotocols. The cultures were subsequently allowed to culture for afurther 5 days culture medium comprising 30 μM ACT 1 peptide (SEQ IDNO:2), 30 μM non-active control peptide (SEQ ID NO:55), or phosphatebuffered saline (PBS) containing no ACT peptide or control peptide. Thenon-active control peptide comprises a polypeptide with a carboxyterminus in which the ACT peptide sequence has been reversed. The aminoterminus of ACT and control peptides are both biotinylated, enablingdetection (i.e., assay) of the peptides in the cell cytoplasm usingstandard microscopic or biochemical methods based on high affinitystreptavidin binding to biotin.

Culture media with added peptides or vehicle control was changed every24 hours during the experiment. FIG. 1a indicates that ACT peptidegreatly increased the extent of Cx43 gap junction formation betweenmyocytes relative to the control conditions (FIGS. 1b and 1c ). As shownin FIG. 4, this increase in Cx43 gap junction formation in response toACT peptide is shared by a number of cell types expressing CX43.

NIH-3T3 cells were grown over 2-3 days until forming a near-confluentmonolayer on a tissue culture dish according to standard protocols andthe monolayer was then pre-treated with ACT 1 peptide (SEQ ID NO:2) for24 hrs, and “scratch-injured” with a p200 pipette tip. The “scratchinjury” was subsequently allowed to repopulate for 24 hours in thepresence of 30 μM ACT 1 peptide (SEQ ID NO:2) dissolved in the culturemedia (FIG. 2a, b ) or in presence of two control conditions (FIG. 2c-f). In the first control condition, the “scratch-injured” cells wereallowed to repopulate for 24 hours in the presence of a non-activecontrol peptide (as in FIG. 1) dissolved in the culture media at aconcentration of 30 μM (FIG. 2c, d ). In the second control condition,phosphate buffered saline (PBS) was added to the culture media and the“scratch-injured” cells were allowed to repopulate in the presence ofthis vehicle control solution containing no ACT peptide or controlpeptide (FIG. 2e, f ). The “scratch injury” of ACT peptide-treated cellsremains relatively repopulated after 24 hours (FIG. 2a ), with few cells(large arrow) repopulating the area within the initial “scratch injury”edges (i.e., within area marked by the small black arrowheads). Bycontrast, in the control conditions in (FIG. 2c, e ), large numbers ofcells (large arrows) have repopulated the area within the initial“scratch injury”. The repopulation of the “scratch injury” occurs inpart via migration of the transformed cells crawling into the “scratchinjury” area. Figures (FIGS. 2b, d, and f ) show proliferating cellnuclear antigen (PCNA) immunolabeling of cells in the “scratch injury”or at the injury edge. ACT peptide treated cells (FIG. 2b ) show onlylow luminosity consistent with background and non-proliferation. Only inthe two control conditions shown in figures (FIG. 2d, f ), are brightlylabeled proliferating cells seen (white arrows). This indicates that theACT peptide has also reduced proliferation of the transformed cells inthis experimental cellular model.

FIG. 3a shows the injury edge of ACT peptide and non-activepeptide-treated control cells at the end of the 24-hour period. Thecells were labeled with fluorescent phalloidin to aid visualization. ACTpeptide-treated cells show low levels of repopulation of the scratchinjury area (white double headed arrows). FIG. 3b shows a bar graph ofthe % area of cells repopulating the scratch injury after 24 hours. Thereduction of cells in the injury area in the presence of ACT peptide isdramatic, with a p value of less than 0.000001.

WB-F344 cells are a transformed rat epithelial cell line derived bytreatment of isolated rat liver cells with a cancer-causing agent (Tsaoet al., 1984; Hayashi et al., 1997; Hayashi et al., 1998; Hayashi etal., 2001). WB-F344 cells were transfected with a cDNA expressionplasmid construct and selected under antibiotic using standard protocolsto generate cell lines that stably expressed anACT-peptide-encoding-polynucleotide (SEQ ID NO:6) operably linked to apromoter sequence or a green fluorescent protein (GFP) polynucleotideoperably linked to a promoter sequence as a control. The polynucleotideencoding the ACT peptide also encoded GFP. As such, expression of theACT peptide could be assayed by standard GFP fluorescence optics on alight microscope. FIG. 4a, b show high magnification images of GFPfluorescence in WB-F344 cell lines expressing GFP alone (FIG. 4a ) orGFP plus the carboxy terminus ACT peptide sequence (FIG. 4a ) or GFPalone (FIG. 4b ). Near confluent monolayers of the WB-F344 cell lineswere “scratch injured” and allowed to repopulate for 24 hours. Similarto the control cases of the NIH-3T3 cells treated with vehicle ornon-active control peptide, the control epithelial cell line expressingGFP repopulated the scratch injury (FIG. 4c ). However, in theepithelial cell line that stably expressed theACT-peptide-encoding-polynucleotide operably linked to a promotersequence, there was inhibited repopulation of the scratch injury (FIG.4d ). In addition to WB-F344 cells lines, NIH-3T3 cell lines have beenmade that stably express an ACT-peptide-encoding-polynucleotide operablylinked to a promoter

Example 2: In Vivo Wound Healing

Neonatal mouse pups were desensitized using hypothermia. A 4 mm longincisional skin injury was made using a scalpel through the entirethickness of the skin (down to the level of the underlying muscle) inthe dorsal mid line between the shoulder blades. 30 μl of a solution of20% pluronic (F-127) gel containing either no (control) or dissolved ACT1 peptide (SEQ ID NO:2) at a concentration of 60 μM was then applied tothe incisional injuries. Pluronic gel has mild surfactant propertiesthat may aid in the uniform dispersion of the ACT peptide in micelles.More importantly, 20% pluronic gel stays liquid at temperatures below15° C., but polymerizes at body temperature (37° C.). This property ofpluronic gel probably aided in the controlled release of peptide intothe tissue at the site of incisional injury, protecting the peptide frombreak-down in the protease-rich environment of the wound and alsoenabling active concentrations of the peptide to maintained overprolonged periods. Control or ACT peptide containing gel was appliedsubsequently 24 hours after the initial application. No furtherapplication of control and ACT peptide containing gel was made after thesecond application. By 48 hours it can be noted that the ACT peptidetreated injury (FIG. 5a ) is significantly more closed, less inflamed,less swollen (note ridges at the wound edge), and generally more healedin appearance than the control injury that received no ACT peptide (FIG.5b ). These differences in inflammation, swelling and healing betweenthe control and ACT peptide and control treated injury persisted at the72 (FIG. 5c, d ) and 96 (FIG. 5e, f ) hour time points. At 7 days, theACT peptide wound (FIG. 5g ), had a smoother and less scarred appearancethan the control peptide-treated injury (FIG. 5h ). Note that images ofthe same injury on the same animal are shown at the different timepoints during the healing time course.

Anesthetized adult mice had 8 mm wide circular excisional skin injuriesmade by fine surgical scissors down to the underlying muscle in thedorsal mid line between the shoulder blades (FIG. 6a, b ). The boundaryof the injury was demarcated by an 8 mm wide circular template cut in aplastic sheet. 100 μl of a solution of 30% pluronic gel containingeither no (control) or dissolved ACT 1 peptide (SEQ ID NO:2) at aconcentration of 100 μM was then applied to the excisional injuries.Control or ACT peptide containing gel was applied subsequently 24 hoursafter the initial application. No further applications of control andACT peptide containing gel were made after the second application. TheACT peptide-treated large excisional injury (FIG. 6 a, c, e, g, i)closed faster, was less inflamed in appearance, healed faster andscarred less than the control injury that received no ACT peptide (FIG.6 b, d, f, h, j) over the 14 day time course. Indeed, the control injuryat 14 days still shows a partial scab indicating that acute healing ofthe injury was incomplete (FIG. 6j ).

Skin biopsies of the entire wound site were taken from some of the 24hours following the excisional injury. These skin samples were fixed in2% paraformaldehyde, paraffin-embedded, sectioned and Hemotoxylin andEosin (H&E) histochemically stained using standard protocols. FIGS. 7aand 7b show low magnification survey views of cross-sections from nearthe center of the wound of ACT peptide and control treated injuries,respectively. The wound edge (marked by the small arrows), bounded byskin of normal histological appearance, can be seen in both cases. Ablack rectangle is placed over the images in FIGS. 7a and 7b at the lefthand wound edge. The histological structures within the black rectangleplaced over the left hand wound edges in FIGS. 7a and 7b are shown athigher magnification in FIGS. 7c and 7d for ACT peptide and controltreated tissues, respectively. Of interest is a “collar-like” tissue ofaligned fibrous material (arrowed) projecting from basal parts of theinjury to or toward the wound edge and exterior surface of injury.Fibrous material serves as a substrate for migration of inflammatorycells moving to the injury surface (Elder et al., 1997). Interestingly,the aligned fibrous substrate has the appearance of being much moreorganized in the control injury (FIG. 7d ) than in the ACT peptidetreated injury (Fig. c). Also, there is a considerably lower density ofinflammatory cells studding the fibrous substrate in the ACTpeptide-treated tissue. This is confirmed in (FIG. 7f ) and (FIG. 7e )where regions of histological section within the black rectangles shownin (FIG. 7d ) and (FIG. 7c ) are respectively shown at highermagnification. The inflammatory cells studding the aligned fibroussubstrate include mast cells, neutrophils and macrophages. Theseinflammatory cells occur at much higher density in the control injurythan in the ACT peptide treated injury.

At the end of the 14 day period, skin biopsies of the entire excisionalinjury were taken and histological sections from these skin samples wereH&E histochemically stained. FIGS. 8a and 8b show low magnificationsurvey views of cross-sections from near the center of the injury of ACTpeptide and control, respectively. The wound edge (marked by the smallarrows), bounded by skin of normal histological appearance, can be seenin both cases. A black rectangle is placed over the images in FIGS. 8aand 8b near the center of each injury. The histological structureswithin these two rectangles are shown at higher magnification in FIGS.8c and 8d for the ACT peptide and control tissues, respectively. It isevident that tissue within the ACT peptide treated injury locus hasconsiderably more complexity. At the external surface of the ACT treatedwound, there is a continuous layer of epithelial cells indicating thatre-epithelization of the injured surface is complete, albeit that theepithelium is as yet relatively thin near the center of the wound (FIG.8c ). Unusually, regenerating hair follicles can already be seendifferentiating de novo from stem cells in the new epithelium coveringthe healed injury (FIG. 8c , small arrows). By comparison,re-epithelization of the injury surface is incomplete and there is nosign of regenerating hair follicles in the epithelium of the controlinjury. Beneath the reformed epithelium of the ACT peptide treatedinjured skin, considerable restoration of normal structural complexityis seen, with glandular structures, fibrous and connective tissues,vascular tissues, muscle and fat cells all in evidence (FIG. 8a, c ). Aswith the hair follicles, this tissue complexity was regenerated bydifferentiation of stem cells. By contrast, in the control injury thewound tissue is completely dominated by a uniform and large plug offibrous scar tissue (FIG. 8b, d ), with other complexity of tissuestructure not particularly in evidence within this scar tissue.

Anesthetized adult mice had 2 small (5 mm diameter) excisional skinwounds made by fine surgical scissors on the neck and (upper) back. Theboundaries of the injuries were demarcated by a 5 mm wide circulartemplate cut in a plastic sheet. 50-60 μl of a solution of 30% pluronicgel containing either no (control) or one of the ACT peptides (ACT 2—SEQID NO:1, ACT 1—SEQ ID NO:2, ACT 3—SEQ ID NO:3, ACT 4—SEQ ID NO:4, ACT5—SEQ ID NO:5) dissolved at concentrations of 100 μM were then appliedto the excisional injuries. Control or ACT peptide-containing gels wereapplied subsequently 24 hours after the initial application. No furtherapplications of control and ACT peptide-containing gel were made afterthe second application. It can be noted in the case of ACT 1 (FIG. 9e-h), ACT 2 (FIG. 9i-l ), ACT 3 (FIG. 9m-p ), and ACT 5 (FIG. 9u-x )peptides that excisional injuries closed faster, were less inflamed inappearance, healed faster and scarred less than the control injury thatreceived no ACT peptide (FIG. 9a-d ) over the 240 hour time course (10days). The ACT 4 peptide (FIG. 9q-t ) also appeared to show modestimprovement in healing over the control during the time course. Notethat the same wound on the same animal is shown at the different timepoints during the healing time course.

The area of open wound was measured during the time course using NIHimage according to standard protocols on multiple (˜5 mice per controlor treatment condition) adult mice. These individual area measurementswere then normalized to (i.e., divided by) the average area measured forthe control injuries for a given time point, multiplied by 100 to give a% of unclosed wound relative to the control and then plotted againsttime. A Mann-Whitney U-test was used to statistically assess the effectsof ACT peptides over the time course. ACT 1, ACT 2, ACT 3, and ACT 5peptides significantly improved wound closure rates following excisionalinjury. These treatments provided results with significant p values. TheACT 1 and ACT 3 quantifiably gave the most pronounced improvements overthe control. A more modest, although consistent, improvement was alsoobserved for the ACT 4 peptide over the control.

Anesthetized adult rats were positioned in a stereotaxic apparatus. Amidline incision was made on the scalp to expose the skull. Astereotaxic drill was sighted 2 mm posterior to the bregma and 2 holeswere drilled with a 1 mm spherical bit, each at 2.5 mm to the right andleft of the bregma, and 3.5 mm below the dura. A cerebral lesion wasmade by inserting an 18-gauge needle. The coordinates were determinedfrom the atlas by Paxinos and Watson (1986). The hollow fiber membrane(HFM) was inserted in the hole and external skin sutures were placed tocover the stab. The ACT peptide was dissolved at 100 μM concentration ina 2% collagen vehicle solution contained within the HFM. Studies ofisolated HFMs indicated that these bioengineered constructs were capableof slow release of detectable levels of ACT peptide (as assayed bybiotin-streptavidin reaction) in aqueous solutions for periods of atleast 7 days. Reactive astrocytosis associated with inflammation andsubsequently with glial scar formation follows a well characterized timecourse after brain injury in rodent models (Norenberg, 1994; Fawcett andAsher, 1999). Typically, the astrocytic response in rat brain peaksafter a week, together with loss of neurons and other aspects of braintissue complexity. Subsequently with the emergence of glial scar tissue,the density of GFAP-positive astrocytes decreases. FIGS. 10b and 10cshow low magnification survey views of sections of brain tissue (cortex)surrounding HFM implants filled with ACT peptide plus vehicle gel orcontrol collagen vehicle gel or ACT peptide plus vehicle gel a weekfollowing brain penetration injury. In the control tissue (FIG. 10c ), ahigh density of immunolabeled GFAP-positive astrocytes is observed nearthe site of injury caused by the HFM. The density of these cells appearsto diminish slightly distal from the injury. By contrast, a much lowerdensity of GFAP-positive astrocytes is observed adjacent the HFM filledwith ACT peptide (FIG. 10b ). Indeed, the levels of GFAP positive cellsare not dissimilar to those seen in normal uninjured brain tissue. Theregions of tissue within the white rectangles in FIGS. 10b and 10c areshown at higher magnification in FIGS. 10d and 10e , respectively. Inthe brain injury treated by ACT peptide (FIG. 10d ), it can be seen thatGFAP-positive astrocytes are not only less numerous, but are alsosmaller than those seen in the control injury (FIG. 10e ).

FIGS. 11a and 11b show low magnification survey views of sections ofbrain tissue (cortex) surrounding HFM implants (implant or injury borderis shown by arrows) filled with control collagen vehicle gel (FIG. 11b )or ACT peptide plus vehicle gel (FIG. 11a ) at 1 week following brainpenetration injury. In the control tissue (FIG. 11b ), a high density ofimmunolabeled GFAP-positive astrocytes and low density of NeuNimmunolabeled neurons are observed near the site of injury caused by theHFM. The density of these cells appears to diminish and increase distalfrom the HFM, respectively. By contrast, a much lower density ofGFAP-positive astrocytes and higher numbers NeuN immunolabeled neuronsare observed proximal (as well as distal) to the HFM filled with ACTpeptide ((FIG. 11a ). The areas in FIGS. 11a and 11b proximal to theHFMs are shown at high magnification views of in FIGS. 11c and 11d ,respectively. Again, in the control tissue (FIG. 11d ) a strikingincrease in the density of GFAP-positive astrocytes and a reduceddensity of NeuN-positive neurons is observed compared to ACT peptidetreated tissueseen (FIG. 11c ). A complementary pattern is observed nearthe HFM containing ACT peptide, with NeuN positive neurons predominatingover astrocytes (FIG. 11c ). Interestingly, the high magnification viewshown in FIG. 11d reveals a high frequency of neurons in the process offission relative to the control (FIG. 11c ). This suggests that the highdensity of neurons associated with ACT peptide treatment may be fromgeneration of new neurons. ACT peptide can also increase neuronaldensity in part by sparing neurons from cell death following braininjury.

Example 3: Treatment of Acute Spinal Cord Injury

Subjects with acute spinal cord injuries represent a seriouslyproblematic group for whom even a small neurological recovery offunction can have a major influence on their subsequent independence. Inone example, a subject with acute spinal cord injury receives a bolusinfusion of a 0.02% to 0.1% solution of ACT peptide (e.g., SEQ ID NO:1)over 15 min within 8 h directly into the site of acute spinal cordinjury, followed 45 min later by an infusion of 0.01% solution of ACTpeptide for a subsequent 23 to 48 hours. In another example, ACT peptideis used to coat slow release nanoparticles loaded within 8 h directlyinto the site of acute spinal cord injury or tissue engineeredbioscaffolds designed to promote neural reconnection across the zone ofacute spinal cord lesion. Improvement in function are assessed by adoctor at intervals (e.g., 6, 12, 26 and 52 weeks) following treatmentby neurological outcome tests including assessments designed to measuremotor activity, pinprick skin sensitivity and recovery of sensation.

Example 4: Quantitative Assessment of Wound Closure, TissueRegeneration, and Tensile Strength of Excisional Skin Wounds

ACT peptide (n=12) and control (n=8) 5 mm-diameter excisional skinwounds were generated on adult mice as described above. Quantitativeassessments of wound closure rate, counts of regenerated hair folliclesand tensile strength measurements were then undertaken on the woundedskin at time points up to 90 days following the initial insult. Relativeto control wounds, closure was significantly enhanced within 24 hours ofpeptide treatment. Similarly, at 10 days, when most wounds were nearingcompletion of closure, a highly significant difference was stillmaintained such that ACT peptide-treated wounds were on average 43%smaller than control wounds. At 10 days ACT peptide wounds showed asignificant 3.2 fold increase in the number of regenerated hairfollicles per unit area of the healed wound over control wounds.

Studies were undertaken of the mechanical properties of healed 5 mmdiameter excisional wounds at 1 month and 3 months following injury. Formechanical property measurements, the skin samples were obtained aftersacrificing the animal and evaluated using a MTS 858 Mini Bionix (MTSSystems Corporation, MN, USA) equipped with a 5 kg load cell. Duringmeasurement the skin sample was extended to break at a rate of 0.5 mm/sForce and extension was measured at break. The tensile strength (stress)and extension to break (strain) was calculated as follows, Stress(N/mm²)=Force at break (N)/cross-sectional area of sample (mm²). Strain(%)=[Increase in length at break (mm)/Original length (mm)]×100. Stressand strain calculations for each wounded skin sample was normalized to anormal skin sample from a nearby area collected from the same animal.

At 1 month, the stress (i.e., normalized force) required to breakwounded skin was similar to that of control wounded skin. At 3 months,normalized stress to break of peptide-treated wound skin was on averagedouble that of control wounded skin, although the high variance withinthe treatment group precluded significant mean separation from thecontrol. This result demonstrates that the intrinsic tensile strength ofpeptide-treated wounds was as good or better than that of untreatedwounds. Further, significant improvements in extensibility ofpeptide-treated wounds were found. The amount of strain (i.e.,extensibility) required to break peptide-treated wounds was modestlyimproved over control wounds at 1 month. At 3 months, peptide-treatedwounds showed a more striking improvement, increasing to a near normal90% of unwounded skin. By contrast, control wounds at 3 months remainedonly 60% as extensible as normal skin.

It is understood that the disclosed method and compositions are notlimited to the particular methodology, protocols, and reagents describedas these may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present invention which willbe limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “apolypeptide” includes a plurality of such polypeptides, reference to“the polypeptide” is a reference to one or more polypeptides andequivalents thereof known to those skilled in the art, and so forth.

“Optional” or “optionally” means that the subsequently described event,circumstance, or material may or may not occur or be present, and thatthe description includes instances where the event, circumstance, ormaterial occurs or is present and instances where it does not occur oris not present.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, also specifically contemplated and considered disclosed isthe range from the one particular value and/or to the other particularvalue unless the context specifically indicates otherwise. Similarly,when values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms another,specifically contemplated embodiment that should be considered disclosedunless the context specifically indicates otherwise. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint unless the context specifically indicates otherwise. Finally,it should be understood that all of the individual values and sub-rangesof values contained within an explicitly disclosed range are alsospecifically contemplated and should be considered disclosed unless thecontext specifically indicates otherwise. The foregoing appliesregardless of whether in particular cases some or all of theseembodiments are explicitly disclosed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present method andcompositions, the particularly useful methods, devices, and materialsare as described. Publications cited herein and the material for whichthey are cited are hereby specifically incorporated by reference.Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such disclosure by virtue of priorinvention. No admission is made that any reference constitutes priorart. The discussion of references states what their authors assert, andapplicants reserve the right to challenge the accuracy and pertinency ofthe cited documents. It will be clearly understood that, although anumber of publications are referred to herein, such reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the method and compositions described herein. Suchequivalents are intended to be encompassed by the following claims.

E. REFERENCES

-   Alonso L, Fuchs E. Stem cells of the skin epithelium. Proc Natl Acad    Sci USA. 2003 Sep. 30; 100 Suppl 1:11830-5, 2003-   Barker R J, Price R L, Gourdie R G. Increased association of ZO-1    with Connexin43 during remodeling of cardiac gap junctions. Circ    Res. February 22; 90(3):317-24 (2002).-   Bucci, M. et al. In vivo delivery of the caveolin-1 scaffolding    domain inhibits nitric oxide synthesis and reduces inflammation.    Nat. Med. 6, 1362-1367 (2000).-   Chien K R. Stem cells: lost in translation. Nature. April 8;    428(6983):607-608 (2004).-   Derossi, D., Joliot, A. H., Chassaing, G. & Prochiantz, A. The third    helix of Antennapedia homeodomain translocates through biological    membranes. J. Biol. 679-686 (2000).-   Elmquist, A., Lindgren, M., Bartfai, T. & Langel, U.    VE-cadherin-derived cell-penetrating peptide, pVEC, with carrier    functions. Exp. Cell Res. 269, 237-244 (2001).-   Elder D., Elenitsas R, Jawaorsky C, & Johnson B. Lever's    histopathology of the skin. Lippincott-Raven Publishers, (1997).-   Fawcett J W, Asher R A. The glial scar and central nervous system    repair. Brain Res. Bull. 49:377-391 (1999).-   Fischer, P. M. et al. Structure-activity relationship of truncated    and substituted analogues of the intracellular delivery vector    Penetratin. J. Pept. Res. 55, 163-172 (2000).-   Frankel, A. D. & Pabo, C. O. Cellular uptake of the Tat protein from    human immunodeficiency virus. Cell 55, 1189-1193 (1988).-   Fu C T, Bechberger J F, Ozog M A, Perbal B, Naus C C. CCN3 (NOV)    interacts with Connexin43 in C6 glioma cells: possible mechanism of    Connexin-mediated growth suppression. J Biol Chem. August 27;    279(35):36943-50 (2004).-   Gao, C. et al. A cell-penetrating peptide from a novel pVII-pIX    phage-displayed random peptide library. Bioorg. Med. Chem. 10,    4057-4065 (2002).-   Giepmans B N. Gap junctions and Connexin-interacting proteins.    Cardiovasc Res. May 1; 62(2):233-45 (2004).-   Goodenough D A, Paul D L. Beyond the gap: functions of unpaired    connexon channels. Nat Rev Mol Cell Biol. April; 4(4):285-94 (2003).-   Green, M. & Loewenstein, P. M. Autonomous functional domains of    chemically synthesized human immunodeficiency virus tat    trans-activator protein. Cell 55, 1179-1188 (1988).-   Hayashi T, Matesic D F, Nomata K, Kang K S, Chang C C, Trosko J E.    Stimulation of cell proliferation and inhibition of gap junctional    intercellular communication by linoleic acid. Cancer Lett.    112:103-111 (1997).-   Hayashi T, Nomata K, Chang C C, Ruch R J, Trosko J E. Cooperative    effects of v-myc and c-Ha-ras oncogenes on gap junctional    intercellular communication and tumorigenicity in rat liver    epithelial cells. Cancer Lett. 128:145-154 (1998).-   Hayashi T, Trosko J E, Hamada K. Inhibition of gap junctional    intercellular communication in rat liver epithelial cells with    transforming RNA. FEBS Lett. 491:200-206 (2001).-   Hong, F. D. & Clayman, G. L. Isolation of a peptide for targeted    drug delivery into human head and neck solid tumors. Cancer Res. 60,    6551-6556 (2000).-   Kajstura J, Rota M, Whang B, Cascapera S, Hosoda T, Bearzi C,    Nurzynska D, Kasahara H, Zias E, Bonafe M, Nadal-Ginard B, Torella    D, Nascimbene A, Quaini F, Urbanek K, Leri A, Anversa P. Bone marrow    cells differentiate in cardiac cell lineages after infarction    independently of cell fusion. Circ Res. January 7; 96(1):127-37    (2005).-   Lin, Y. Z., Yao, S. Y., Veach, R. A., Torgerson, T. R. & Hawiger, J.    Inhibition of nuclear translocation of transcription factor NF-κB by    a synthetic peptide containing a cell membrane-permeable motif and    nuclear localization sequence. J. Biol. Chem. 270, 14255-14258    (1995).-   Lundberg, P. et al. Cell membrane translocation of the N-terminal    (1-28) part of the prion protein. Biochem. Biophys. Res. Commun.    299, 85-90 (2002).-   Matsushita M, Noguchi H, Lu Y F, Tomizawa K, Michiue H, Li S T,    Hirose K, Bonner-Weir S, Matsui H. Photo-acceleration of protein    release from endosome in the protein transduction system. FEBS Lett.    13; 572(1-3):221-6. (2004).-   Morris, M. C., Depollier, J., Mery, J., Heitz, F. & Divita, G. A    peptide carrier for the delivery of biologically active proteins    into mammalian cells. Nature Biotechnol. 19, 1173-1176 (2001).-   Norenberg M D. Astrocyte responses to CNS injury. J. Neuropathol.    Exp. Neurol. 53:213-220 (1994).-   Oehlke, J. et al. Cellular uptake of an α-helical amphipathic model    peptide with the potential to deliver polar compounds into the cell    interior non-endocytically. Biochim. Biophys. Acta. 1414, 127-139    (1998).-   Park, C. B., Yi, K. S., Matsuzaki, K., Kim, M. S. & Kim, S. C.    Structure-activity analysis of buforin II, a histone H2A-derived    antimicrobial peptide: the proline hinge is responsible for the    cell-penetrating ability of buforin II. Proc. Natl Acad. Sci. USA    97, 8245-8250 (2000).-   Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. 2nd    ed. San Diego, Calif.: Academic; 1986.-   Pich A, Chiusa L, Navone R. Prognostic relevance of cell    proliferation in head and neck tumors Annals of Oncology 2004    15(9):1319-1329.-   Pooga, M., Hallbrink, M., Zorko, M. & Langel, U. Cell penetration by    transportan. FASEB J. 12, 67-77 (1998).-   Poss K D, Wilson L G, Keating M T. Heart regeneration in zebrafish.    Science. December 13; 298(5601):2188-90 (2002).-   Rousselle, C. et al. New advances in the transport of doxorubicin    through the blood-brain barrier by a peptide vector-mediated    strategy. Mol. Pharmacol. 57(4):679-86 (2000).-   Sawada, M., Hayes, P. & Matsuyama, S. Cytoprotective    membrane-permeable peptides designed from the Bax-binding domain of    Ku70. Nature Cell Biol. 5, 352-357 (2003).-   Silver J, Miller J H. Regeneration beyond the glial scar. Nat Rev    Neurosci. February; 5(2):146-56 (2004).-   Songyang, Z. et al. Recognition of unique carboxyl-terminal motifs    by distinct PDZ domains. Science 275, 73-77 (1997).-   Tsao M S, Smith J D, Nelson K G, Grisham J W. A diploid epithelial    cell line from normal adult rat liver with phenotypic properties of    ‘oval’ cells. Exp. Cell Res. 154:38-52 (1984).-   Vigneron, J. P. et al. Guanidinium-cholesterol cationic lipids:    Efficient vectors for the transfection of eukaryotic cells. Proc.    Natl. Acad. Sci. USA. 93, 9682-9686 (1998).-   Wadia J S, Stan R V, Dowdy S F. Transducible TAT-HA fusogenic    peptide enhances escape of TAT-fusion proteins after lipid raft    macropinocytosis. Nat Med. 10(3):310-5. (2004).-   Ming Y. W. Chu^(a), Milton H. Lipsky^(a), Lorrin K. Yee^(a,c), John    Epstein^(a), Katharine A. Whartenby^(a), Scott Freeman^(e), Tian M.    Chen^(a), Edward Chu^(c,d), Edwin N. Forman^(b), Paul Calabresi^(a)    Predictive Sensitivity of Human Cancer Cells in vivo Using    Semipermeable Polysulfone Fibers Pharmacology 1998; 56:318-326-   Orlandini G C, Margaria R. Evaluation of the efficiency of a new    hollow fiber plasmapheresis filter. Int J Artif Organs. 1983 Jul.; 6    Suppl 1:103-6.-   Wilgus T A, Vodovotz Y, Vittadini E, Clubbs E A, Oberysztn T M.    Reduction of scar formation in full-thickness wounds with topical    celecoxib treatment. Wound Rep Reg 2003; 11:25-34.-   Yoo D S. The dielectric properties of cancerous tissues in a nude    mouse xenograft model. Bioelectromagnetics. 2004 Oct.; 25(7):492-7.

SEQUENCES

(ACT 2) SEQ ID NO: 1 PSSRASSRASSRPRPDDLEI (ACT 1) SEQ ID NO: 2 RPRPDDLEI(ACT 3) SEQ ID NO: 3 RPRPDDLEV (ACT 4) SEQ ID NO: 4 RPRPDDVPV (ACT 5)SEQ ID NO: 5 KARSDDLSV SEQ ID NO: 6 aga cct cgg cct gat gac ctg gag att(Antp) SEQ ID NO: 7 RQPKIWFPNRRKPWKK (Antp/ACT 2) SEQ ID NO: 8RQPKIWFPNRRKPWKKPSSRASSRASSRPRPDDLEI (Antp/ACT 1) SEQ ID NO: 9RQPKIWFPNRRKPWKKRPRPDDLEI (Antp/ACT 3) SEQ ID NO: 10RQPKIWFPNRRKPWKKRPRPDDLEV (Antp/ACT 4) SEQ ID NO: 11RQPKIWFPNRRKPWKKRPRPDDVPV (Antp/ACT 5) SEQ ID NO: 12RQPKIWFPNRRKPWKKKARSDDLSV (encodes polypeptide of SEQ ID NO 9)SEQ ID NO: 13 cgg cag ccc aag atc tgg ttc ccc aac cgg cgg aag ccctgg aag aag cgg ccc ggc ccg acg acc tgg aga tc (HIV-Tat) SEQ ID NO: 14GRKKRRQRPPQ (Penetratin) SEQ ID NO: 15 RQIKIWFQNRRMKWKK (Antp-3A)SEQ ID NO: 16 RQIAIWFQNRRMKVVAA (Tat) SEQ ID NO: 17 RKKRRQRRR(Buforin II) SEQ ID NO: 18 TRSSRAGLQFPVGRVHRLLRK (Transportan)SEQ ID NO: 19 GWTLNSAGYLLGKINKALAALAKKIL (model amphipathic peptide)SEQ ID NO: 20 KLALKLALKALKAALKLA (K-FGF) SEQ ID NO: 21 AAVALLPAVLLALLAP(Ku70) SEQ ID NO: 22 VPMLK-PMLKE (Prion) SEQ ID NO: 23MANLGYWLLALFVTMWTDVGLCKKRPKP (pVEC) SEQ ID NO: 24 LLIILRRRIRKQAHAHSK(Pep-1) SEQ ID NO: 25 KETWWETWWTEWSQPKKKRKV (SynB1) SEQ ID NO: 26RGGRLSYSRRRFSTSTGR (Pep-7) SEQ ID NO: 27 SDLWEMMMVSLACQY (HN-1)SEQ ID NO: 28 TSPLNIHNGQKL (Chick alpha Cx43 ACT) SEQ ID NO: 29PSRASSRASSRPRPDDLEI (Human alpha Cx45) SEQ ID NO: 30GSNKSTASSKSPDPKNSVWI (Chick alpha Cx45) SEQ ID NO: 31GSNKSSASSKSGDGKNSVWI (Human alpha Cx46) SEQ ID: 32 GRASKASRASSGRARPEDLAI(Human alpha Cx46.6) SEQ ID: 33 GSASSRDGKTVWI (Chimp alpha Cx36)SEQ ID NO: 34 PRVSVPNFGRTQSSDSAYV (Chick alpha Cx36) SEQ ID NO: 35PRMSMPNFGRTQSSDSAYV (Human alpha Cx47) SEQ ID NO: 36PRAGSEKGSASSRDGKTTW/1 (Human alpha Cx40) SEQ ID NO: 37GYHSDKRRLSKASSKARSDDLSV (Human alpha Cx50) SEQ ID NO: 38PLSRLSKASSRARSDDLTV (Human alpha Cx59) SEQ ID NO: 39PNHVVSLTNNLIGRRVPTDLQI (Rat alpha Cx33) SEQ ID NO: 40PSCVSSSAVLTTICSSDQVVPVGLSSFYM (Sheep alpha Cx44) SEQ ID NO: 41GRSSKASKSSGGRARAADLAI (Human beta Cx26) SEQ ID NO: 42 LCYLLIRYCSGKSKKPV(Human alpha Cx37) SEQ ID: 43 G QK PP SRPS SSAS K KQ*YV(conservative Cx43 variant) SEQ ID 44: SSRASSRASSRPRPDDLEV(conservative Cx43 variant) SEQ ID 45: RPKPDDLEI,(conservative Cx43 variant) SEQ ID 46: SSRASSRASSRPKPDDLEI,(conservative Cx43 variant) SEQ ID 47: RPKPDDLDI(conservative Cx43 variant) SEQ ID 48: SSRASSRASSRPRPDDLDI(conservative Cx43 variant) SEQ ID 49: SSRASTRASSRPRPDDLEI(conservative Cx43 variant) SEQ ID 50: RPRPEDLEI(conservative Cx43 variant) SEQ ID 51: SSRASSRASSRPRPEDLEI,(conservative Cx45 variant) SEQ ID 52: GDGKNSVWV(conservative Cx45 variant) SEQ ID 53: SKAGSNKSTASSKSGDGKNSVWV(conservative Cx37 variant) SEQ ID 54: GQKPPSRPSSSASKKLYV(non-active control peptide) SEQ ID NO: 55 RQPKIWFPNRRKPWKIELDDPRPR(HIV-Tat/ACT 1) SEQ ID NO: 56 GRKKRRQRPPQ RPRPDDLEI (Penetratin/ACT 1)SEQ ID NO: 57 RQIKIWFQNRRMKWKK RPRPDDLEI (Antp-3A/ACT 1) SEQ ID NO: 58RQIAIWFQNRRMKWAA RPRPDDLEI (Tat/ACT 1) SEQ ID NO: 59 RKKRRQRRR RPRPDDLEI(Buforin II/ACT 1) SEQ ID NO: 60 TRSSRAGLQFPVGRVHRLLRK RPRPDDLEI(Transportan/ACT 1) SEQ ID NO: 61 GWTLNSAGYLLGKINKALAALAKKIL RPRPDDLEI(MAP/ACT 1) SEQ ID NO: 62 KLALKLALKALKAALKLA RPRPDDLEI (K-FGF/ACT 1)SEQ ID NO: 63 AAVALLPAVLLALLAP RPRPDDLEI (Ku70/ACT 1) SEQ ID NO: 64VPMLKPMLKE RPRPDDLEI (Prion/ACT 1) SEQ ID NO: 65MAN LGYWLLALFVTMWTDVGLCKKRPKP RPRPDDLEI (pVEC/ACT 1) SEQ ID NO: 66LLIILRRRIRKQAHAHSK RPRPDDLEI (Pep-1/ACT 1) SEQ ID NO: 67KETWWETWWTEWSQPKKKRKV RPRPDDLEI (SynB1/ACT 1) SEQ ID NO: 68RGGRLSYSRRRFSTSTGR RPRPDDLEI (Pep-7/ACT 1) SEQ ID NO: 69SDLWEMMMVSLACQY RPRPDDLEI (HN-1/ACT 1) SEQ ID NO: 70TSPLNIHNGQKL RPRPDDLEI (20 to 120 residues flanking amino acid 363 of human Cx43) SEQ ID NO: 72KGKSDPYHATSGALSPAKDCGSQKYAYFNGCSSPTAPLSPMSPPGYKLVTGDRNNSSCRNYNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFPDD NQNSKKLAAGHELQPLAIVD(20 to 120 residues flanking amino acid 362 of  chick Cx43)SEQ ID NO: 73 KTDPYSHSGTMSPSKDCGSPKYAYYNGCSSPTAPLSPMSPPGYKLVTGDRNNSSCRNYNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFADEHQN TKKLASGHELQPLTIVDQRP(20 to 120 residues flanking amino acid 377 of  human Cx45)SEQ ID NO: 74 LGFGTIRDSLNSKRRELEDPGAYNYPFTWNTPSAPPGYNIAVKPDQIQYTELSNAKIAYKQNKANTAQEQQYGSHEENLPADLEALQREIRMAQERLDLA VQAYSHQNNPHGPREKKAKV(20 to 120 residues flanking amino acid 375 of  chick Cx45)SEQ ID NO: 75 GFGTIRDTLNNKRKELEDSGTYNYPFTWNTPSAPPGYNIAVKPDQMQYTELSNAKMAYKQNKANIAQEQQYGSNEENIPADLENLQREIKVAQERLDMAI QAYNNQNNPGSSSREKKSKA.(20 to 120 residues flanking amino acid 313 of  human Cx37)SEQ ID NO: 76 PYLVDCFVSRPTEKTIFIIFMLVVGLISLVLNLLELVHLLCRCLSRGMRARQGQDAPPTQGTSSDPYTDQVFFYLPVGQGPSSPPCPTYNGLSSSEQNWA NLTTEERLASSRPPLFLDPP(20 to 120 residues flanking amino acid 258 of  rat Cx33) SEQ ID NO: 77CGSKEHGNRKMRGRLLLTYMASIFFKSVFEVAFLLIQWYLYGFTLSAVYICEQSPCPHRVDCFLSRPTEKTIFILFMLVVSMVSFVLNVIELFYVLFKAI KNHLGNEKEEVYCNPVELQK.(enhanced green fluorescent protein) SEQ ID NO: 78MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (ACT 2) SEQ ID NO: 79CCCTCCTCCCGGGCCTCCTCCCGGGCCTCCTCCCGGCCCCGGCCCGAC  GACCTGGAGATC (ACT 1)SEQ ID NO: 80 CGGCCCCGGCCCGACGACCTGGAGATC (ACT 3) SEQ ID NO: 81CGGCCCCGGCCCGACGACCTGGAGGTG (ACT 4) SEQ ID NO: 82CGGCCCCGGCCCGACGACGTGCCCGTG (ACT 5) SEQ ID NO: 83AAGGCCCGGTCCGACGACCTGTCCGTG (Antp) SEQ ID NO: 84CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAG AAG (Antp/ACT 2)SEQ ID NO: 85 CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGCCCTCCTCCCGGGCCTCCTCCCGGGCCTCCTCCCGGCCCCGGCCCGACGACC TGGAGATC (Antp/ACT 1)SEQ ID NO: 86 CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGCGGCCCCGGCCCGACGACCTGGAGATC (Antp/ACT 3) SEQ ID NO: 87CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGCGGCCCCGGCCCGACGACCTGGAGGTG (Antp/ACT 4) SEQ ID NO: 88CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGCGGCCCCGGCCCGACGACGTGCCCGTG (Antp/ACT 5) SEQ ID NO: 89CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGAAGGCCCGGTCCGACGACCTGTCCGTG (Zebrafish alpha Cx43) SEQ ID NO: 90PCSRASSRMSSRARPDDLDV (Chick alpha Cx36) SEQ ID NO: 91PRVSVPNFGRTQSSDSAYV

We claim:
 1. A method of treating radiation injury in a subject,comprising administering to the subject a composition comprising anisolated polypeptide consisting of the carboxy terminal-most 4 to 30contiguous amino acids of an alpha Connexin, or a conservative variantthereof, wherein the composition is administered to the subject in atherapeutically effective amount for the treatment of radiation injury.2. The method of claim 1, wherein the polypeptide consists of thecarboxy terminal-most 5 to 19 contiguous amino acids of the alphaConnexin.
 3. The method of claim 1, wherein the alpha Connexin isConnexin 37, Connexin 40, Connexin 43, or Connexin
 45. 4. The method ofclaim 3, wherein the polypeptide comprises the amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, and SEQ ID NO:5.
 5. The method of claim 4, whereinthe polypeptide comprises the amino sequence of SEQ ID NO:2.
 6. Themethod of claim 1, wherein the polypeptide comprises an amino acidsequence with at least 65% sequence identity to the c-terminal most 9amino acids of SEQ ID NO:1.
 7. The method of claim 1, wherein thepolypeptide comprises an amino acid sequence with at least 75% sequenceidentity to the c-terminal most 9 amino acids of SEQ ID NO:1.
 8. Themethod of claim 1, wherein the polypeptide comprises an amino acidsequence with at least 85% sequence identity to the c-terminal most 9amino acids of SEQ ID NO:1.
 9. The method of claim 1, wherein thecomposition further comprises a cellular internalization sequence. 10.The method of claim 9, wherein the cellular internalization sequencecomprises an amino acid sequence of a protein selected from a groupconsisting of Antennapedia, TAT, HIV-Tat, Penetratin, Antp-3A (Antpmutant), Buforin II, Transportan, MAP (model amphipathic peptide),K-FGF, Ku70, Prion, pVEC, Pep-1, SynB 1, Pep-7, HN-1, BGSC(Bis-Guanidinium-Spermidine-Cholesterol) and BGTC(Bis-Guanidinium-Tren-Cholesterol).
 11. The method of claim 10, whereinthe cellular internalization sequence is Antennapedia, and wherein thesequence comprises the amino acid sequence of SEQ ID NO:7.
 12. Themethod of claim 1, wherein the polypeptide is linked at its aminoterminus to the cellular internalization transporter sequence, andwherein the amino acid sequence of the polypeptide and cellulartransporter sequence is selected from the group consisting of SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.
 13. Themethod of claim 1, wherein the polypeptide comprises a type II PDZbinding motif.
 14. The method of claim 1, wherein the radiation injuryis present in a tissue selected from the group consisting of skin,heart, bone, brain, spinal cord, retina, and peripheral nerve.
 15. Themethod of claim 1, wherein the composition promotes healing.
 16. Themethod of claim 1, wherein the composition reduces inflammation.
 17. Themethod of claim 1, wherein the composition reduces scar formation. 18.The method of claim 1, wherein the composition promotes tissueregeneration.
 19. The method of claim 1, wherein the composition isadministered topically, orally, extracorporeally, intracranially,intravaginally, intraanally, subcutaneously, intradermally,intracardiac, intragastric, intravenously, intramuscularly, byintraperitoneal injection, transdermally, intranasally, or by inhalant.20. The method of claim 1, wherein the composition is appliedimmediately after the injury.